Protocol to detect telomerase activity in adult mouse hippocampal neural progenitor cells using the telomeric repeat amplification protocol assay

Summary

Changes in telomerase activity and telomere length contribute to aging-related decline. Investigating telomerase in aging models provides insights into related pathologies. Here, we present a protocol to detect telomerase activity in adult mouse hippocampal neural progenitor cells using the telomeric repeat amplification protocol assay. We describe steps for isolating and expanding aged mouse hippocampal neural progenitor cells (NPCs) and assessing telomerase using a non-radioactive technique. The protocol emphasizes the significance of understanding telomerase activity in NPCs for neurogenesis and age-related diseases.

Graphical abstract

Highlights

• •Isolation of brain neural progenitor cells from aged mice hippocampus
• •Dissociation of multiple hippocampus tissues into single-cell suspension without additional steps
• •Validated telomerase activity detection strategy for neural progenitor cells from aged mice

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Changes in telomerase activity and telomere length contribute to aging-related decline. Investigating telomerase in aging models provides insights into related pathologies. Here, we present a protocol to detect telomerase activity in adult mouse hippocampal neural progenitor cells using the telomeric repeat amplification protocol assay. We describe steps for isolating and expanding aged mouse hippocampal neural progenitor cells (NPCs) and assessing telomerase using a non-radioactive technique. The protocol emphasizes the significance of understanding telomerase activity in NPCs for neurogenesis and age-related diseases.

Before you begin

Telomerase, a ribonucleoprotein enzyme complex, is vital for maintaining telomere length and genomic stability, impacting cellular aging and proliferation.¹ With age, changes in telomere length and telomerase activity contribute to physiological decline and age-related diseases, making the investigation of telomerase activity in aging models crucial for understanding aging-associated pathologies.²

The Telomeric Repeat Amplification Protocol (TRAP) assay is a powerful tool for detecting and quantifying telomerase activity by amplifying telomeric repeats through PCR. This study aims to detect telomerase activity in neural progenitor cells (NPCs) extracted from the hippocampus of old mice, shedding light on its role in neurogenesis and its implications for aging and neurodegenerative diseases.³

This research introduces a procedure to isolate NPCs from mice aged 30–40 weeks, enabling the assessment of telomerase activity within this age range. Previous studies have isolated NPCs from adult mice, but self-renewal ability in resulting neurospheres was limited.⁴ Babu et al. made a significant advancement by isolating multipotent neural cells from the dentate gyrus (DG) of adult mice, demonstrating their ability to maintain self-renewal and multipotency in monolayer culture. However, this protocol required six mice per cell isolation, posing practical challenges for studies with limited animal availability.⁵ Our protocol overcomes this by enabling the isolation of multipotent NPCs from aged mice using fewer animals and eliminating the need for additional separation steps.

Several detection methods have been developed for TRAP.^6,7 Recently, nonradioactive methods have been introduced to eliminate the use of radioisotopes when labeling the telomerase substrate (TS) primer. Another study detailed the measurement of telomerase activity in samples from various brain regions of mice. However, this approach was applied to mice aged 8–12 weeks, lacking internal controls to validate reaction specificity.⁸ The TRAP assay employed in this study involves a two-step process, allowing for the sensitive and quantitative assessment of telomerase activity in NPCs through gel electrophoresis and real-time PCR analysis. Additionally, it includes internal controls to monitor PCR inhibition.

Ensure that the experimental procedures involving animals are approved by the Institutional Animal Care and Use Committee (IACUC) or an equivalent regulatory body.

Sterilize all laboratory equipment, including microcentrifuge tubes, pipettes, and petri dishes, using appropriate methods such as autoclaving or chemical disinfection. Maintain sterile conditions throughout the experiment to minimize the risk of microbial contamination, which could compromise cell viability and assay accuracy.

Review the experimental protocols and procedures outlined in the study to familiarize yourself with the workflow and critical steps. Verify that you have access to all necessary protocols, including microdissection techniques, cell isolation procedures, TRAP assay protocols, and gel electrophoresis methods.

Ensure that the laboratory workspace is clean and organized to minimize the risk of contamination. Set up necessary equipment, including microcentrifuges, pipettes, thermal cyclers, gel electrophoresis apparatus, and a UV transilluminator.

Prepare all reagents and solutions required for the experiment according to the specified protocols. This includes buffers, media, and enzyme solutions necessary for tissue dissociation, cell lysis, and telomerase activity detection. Use high-quality reagents and nuclease-free water to prevent contamination and ensure assay reliability.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Chemicals, peptides, and recombinant proteins
DMEM/F-12	Thermo Fisher Scientific	10565018
hBFGF	Sigma	F0291
EGF	Sigma	E4127
Accutase	STEMCELL Technologies	07920
Neurobasal A	Thermo Fisher Scientific	10888022
B27 supplement	Thermo Fisher Scientific	12587010
Bradford protein assay kit	Bio-Rad	-
RNaseZAP	Thermo Fisher Scientific	AM9780
50× dNTP mix	Thermo Fisher Scientific	R0192
Titanium Taq DNA polymerase	Scientifix	639208
10× TBE	Bio-Rad	1610770
TEMED (N,N,N′,N′-tetramethylethylenediamine)	Bio-Rad	1610800
10% (wt/vol) Ammonium persulfate solution	Bio-Rad	1610700
Acrylamide (19:1 acrylamide:bis-acrylamide)	Bio-Rad	1610144
GelRed nucleic acid gel stain	Assay Matrix	41003
Glucose	Sigma	G8769
HBSS	Thermo Fisher Scientific	14065056
BSA	Sigma	A4503
EBSS	Thermo Fisher Scientific	14155063
Trypsin	Sigma	T4665
Hyaluronidase	Sigma	H3884
Penicillin/Streptomycin	Thermo Fisher Scientific	15140122
DMSO	Sigma	D8418
MgCl2	Sigma	M8266
PMSF	Sigma	10837091001
2-Mercaptoethanol	Sigma	M3148
Glycerol	Sigma	G5516
Bromophenol blue	Sigma	B0126
EGTA	Sigma	324626
Chaps	Sigma	220201
Powerup Sybr Green master mix	Thermo Fisher Scientific	A25742
HEPES (1 M)	Thermo Fisher Scientific	15630
GlutaMAX	Thermo Fisher Scientific	35050061
Liquid nitrogen	-	-
Experimental models: Cell lines
HeLa cell line	-	-
Experimental models: Organisms/strains
Mice, C57BL/6J	-	-
Oligonucleotides
TS primer, HPLC-purified (5′-AATCCGTCGAGCAGAGTT-3′)	This paper	-
ACX primer, HPLC-purified (5′-GCGCGG CTTACCCTTACCCTTACCCTAACC-3′)	This paper	-
NT primer, HPLC-purified (5′-ATCGCTTCTCGGCCTTTT-3′)	This paper	-
TSNT primer, HPLC-purified (5′-AATCCGTCGA GCAGAGTTAAAAGGCCGAGAAGCGAT-3′)	This paper	-
Other
Pasteur pipettes	Eppendorf	-
Dissecting tools	-	-
Tissue culture hood	-	-
CO2 incubator set to 5% CO2 and 37°C	-	-
Tabletop centrifuge	Thermo Fisher Scientific	-
Inverted microscope	-	-
Thermocycler	LC 480, Roche	-
Heat block at 85°C	Thermo Fisher Scientific	-
Polyacrylamide vertical gel electrophoresis apparatus	Bio-Rad	-
Power supply	Bio-Rad	-
Rocker	-	-
Phosphorimager	ChemiDoc imaging system, Bio-Rad	-

Materials and equipment

EGF-Stock with a concentration of 10 μg/mL

Reagent	Final concentration	Amount
EGF	N/A	100 μg
DMEM: F12	N/A	10 mL
Glucose	4.5%	100 μL
Penicillin/Streptomycin	N/A	100 μL

Note: Make 100 μL aliquots and store at −20°C.

FGF-Stock with a concentration of 10 μg/mL

Reagent	Final concentration	Amount
FGF	N/A	10 μg
DMEM: F12	N/A	10 mL
Glucose	4.5%	100 μL
Penicillin/Streptomycin	N/A	100 μL

Note: Make 100 μL aliquots and store at −20°C.

Solution A (HBSS-Glucose)

Reagent	Final concentration	Amount
HBSS	N/A	50 mL
D-Glucose	N/A	9.0 mL
HEPES	N/A	7.5 mL
ddH2O	N/A	433.5 mL
Total	N/A	500 mL

Note: Adjust pH to 7.5. Make aliquots and store at −20°C.

Solution B (Sucrose-HBSS)

Reagent	Final concentration	Amount
HBSS	N/A	25 mL
Sucrose	N/A	154 g
ddH2O	N/A	475 mL
Total	N/A	500 mL

Note: Adjust pH to 7.5. Make aliquots and store at −20°C

Solution C (BSA-EBSS-HEPES)

Reagent	Final concentration	Amount
BSA	N/A	20 g
HEPES	N/A	10 mL
EBSS	N/A	490 mL
Total	N/A	500 mL

Note: Adjust pH to 7.5. Make aliquots and store at −20°C.

• •Dissociation Media: Solution A (10 mL), Trypsin (13.3 mg), Hyaluronidase (7.0 mg). Make aliquots and store at −20°C.

Adult Growth Medium

Reagent	Final concentration	Amount
Penicillin/Streptomycin	100 units/mL	500 μL
HEPES	8 mM	400 μL
B27	N/A	1000 μL
FGF∗	10 ng/mL	50 μL
EGF∗	10 ng/mL	50 μL
DMEM: F12/Glutamax	N/A	Fill up to 50 mL (Total volume)

Note: Store at 4°C up to 1 month. ∗Add freshly just before use.

• •Cryoprotectant medium: Adult Growth Medium with 10% of DMSO. Make fresh every time.
• •Poly-L-ornithine solution: Dilute the stock solution into sterile Milli-Q H₂O to make a final concentration of 10 μg/mL. This solution is stable at 4°C for 2–4 weeks.
• •Laminin solution: Dilute laminin in sterile PBS to make a final concentration of 5 μg/mL. Aliquot and store at −20°C.

NPC Differentiation media

Reagent	Final concentration	Amount
Glutamax	N/A	500 μL
Penicillin/Streptomycin	100 units/mL	500 μL
B27	N/A	1000 μL
Neurobasal A Medium	N/A	Fill up to 50 mL (Total volume)

Note: Store at 4°C up to 1 month.

• •50× TRAP primer mix recipe: The primer mix comprises the substrate for the 36-bp internal standard control (TSNT) and reverse primers (NT and ACX) required for amplifying both the internal standard control (IC) and telomerase products. To prepare, dilute the TSNT oligonucleotide to a concentration of 100 μM using RNase/DNase-free H₂O. Next, combine ACX and NT primers with RNase/DNase-free H₂O (resulting in a final concentration of 100 ng/μL). Finally, add TSNT to the above mixture (final concentration of 0.01 × 10⁻¹⁸ mol/μL). Aliquot the mixture and store it at −20°C.

CRITICAL: Utilize filter tips during the preparation of this recipe and ensure thorough cleaning of the workspace with bleach afterward to eliminate any traces of the mixture. This step is crucial to prevent contamination during future TRAP assays.

1× lysis buffer

Reagent	Final concentration	Amount
Tris–HCl	10 mM	0.5 mL
MgCl2	1 mM	0.05 mL
EGTA	1 mM	0.05 mL
PMSF	0.1 mM	0.005 mL
2-mercaptoethanol	5 mM	0.25 mL
Chaps	0.5%	0.25 mL
Glycerol	10%	5 mL
ddH2O	N/A	43.95 mL
Total	N/A	50 mL

Note: Add RNase inhibitor just before use. Store at 4°C.

10× TRAP buffer

Reagent	Final concentration	Amount
Tris-HCl, pH 8.3	200 mM	2 mL
MgCl2	15 mM	0.15 mL
KCl	630 mM	6.3 mL
Tween 20	0.5%	0.05 mL
EGTA	10 mM	0.1 mL
ddH2O	N/A	1.4 mL
Total	N/A	10 mL

Note: Store at 4°C.

• •6× gel-loading dye: 0.25% (wt/vol) bromophenol blue in 50% (vol/vol) glycerol/50 mM EDTA. Store at 4°C.

CRITICAL: The preparation of in-house gel-loading dye was essential in our experiment to generate accurate TRAP ladder bands.

Step-by-step method details

Isolation of neural progenitor cells from adult mouse hippocampus brain

Timing: 45–60 min (for step 1)

Timing: 1–2 h (for step 2)

Timing: 30–40 min (for step 3)

This section outlines a detailed protocol for extracting and purifying neural progenitor cells from the hippocampus of adult mice. This procedure enables researchers to obtain a highly enriched population of NPCs, facilitating studies on neurogenesis and related processes in the adult brain.

• 1.
  Microdissection of hippocampus from adult mouse brain.

  • a.
    Humanely euthanize adult mice aged 30–40 weeks following an Institutional Animal Care and Use Committee-approved protocol.
  • b.
    Place the mice into the CO2 chamber, slowly introducing carbon dioxide to induce a gradual state of anesthesia and unconsciousness, ultimately followed by euthanasia through decapitation.
  • c.
    Using large scissors, remove the head just above the cervical spinal cord region.
  • d.
    Afterward, employ small, pointed scissors to make a medial caudal-rostral cut and remove the head’s skin.
  • e.
    Perform a longitudinal incision at the base of the skull using small scissors and continue cutting along the sagittal suture.
  • f.
    Then, use pointed forceps to grasp each hemisphere’s skull at the incision’s base, peeling outward to expose the brain.
  • g.
    Repeat for the other hemisphere.
  • h.
    Invert the animal’s head, cut the optic nerves while allowing the brain to slip into a 50-mL Falcon tube containing 20 mL of ice cold HBSS.

    CRITICAL: Maintain the brain in cold Solution A on ice during subsequent dissection.

  • i.
    Transfer the brain from the 50-mL tube to a 10-cm petri dish with 20 mL of cold Solution A.
  • j.
    Place a small piece of filter paper, slightly moisten it with a wet sterile swab, and then position the brain onto the wet filter paper using curved-pointed forceps.
  • k.
    Proceed to chop the brain into coronal sections using a sterile scalpel, using a wet sterile swab to collect hippocampus tissue into a 6-cm petri dish filled with 5 mL of Solution A.

    CRITICAL: Keep the petri dish containing brain sections and solution A on ice during the chopping and dissection period.

• 2.
  Isolation of neural progenitor cells.

  • a.
    After finishing the dissection of all the brain sections, spin down the tissue chunks in a low-speed centrifuge at 200 g for 1 min at room temperature (20–25°C).
  • b.
    In the TC hood remove the supernatant and add 5 mL of Dissociation Media to each 15 mL conical tubes containing tissue chunks. Place the conical tubes into the incubator at 37°C for 15 min.

    CRITICAL: Do not incubate longer than 30 min, as this will decrease the viability of the cells.

  • c.
    Pre-wet a fire-polished Pasteur pipette to triturate tissue by pipetting up and down 10-20 times until there is no tissue clump, and then incubate again for 15 min at 37°C.

    CRITICAL: Avoid generating air bubbles when triturating the tissue, as this will reduce the viability of the cells.

    CRITICAL: It is important to dissociate the hippocampus into single cells, as any remaining aggregates can result in reduced yield.

  • d.
    Add 1 volume (5 mL) of ice-cold Solution C to the tube to inactivate the Trypsin. Pipette cells up and down using a 10 mL Pasteur pipette.
  • e.
    Pass cells through a 70 μm strainer into a 50 mL Falcon Tube and then centrifuge the cells down for 5 min at 200 g at 4°C in a centrifuge.
  • f.
    Remove supernatant and resuspend cells in 10 mL ice cold Solution B using a 10 mL plastic pipette.

    CRITICAL: Gently remove the supernatant, as the pellet may not be firmly attached to the bottom of the tube.

  • g.
    Spin the cells down for 10 min at 200 g at 4°C and then add 10 mL of Solution C into a new 15 mL Falcon Tube.
  • h.
    Remove supernatant from the centrifuged cells and resuspend them in 2 mL ice-cold Solution C.
  • i.
    After complete resuspension, add the 2 mL to the other 10 mL.
  • j.
    Centrifuge cells down for 7 min at 200 g at 4°C and re-suspend the cell pellet in 5 mL of adult growth medium (AGM).
  • k.
    Then plate the cells into a well of a 24-well tissue culture plate.
  • l.
    Incubate cells in the CO₂ incubator for 48 h at 37°C.
  • m.
    Change half the AGM, avoiding removing any cells.
  • n.
    Continue to incubate cells for 7–14 days, changing half the AGM, adding EGF and FGF (final concentration: 10 ng/ml) every other day and monitoring the cells for the formation of neural spheres. Neural spheres should form in cultures in 1–2 weeks.

    Note: It is imperative to prevent the excessive formation of spheres, as this may result in cell loss, due to diminished access to nutrients and oxygen.

    Note: We observed that the hippocampi from three animals, totaling six, can be resuspended in 5 mL of AGM and then seeded into a small culture flask.

• 3.
  Passage of neural progenitor cells into an adherent monolayer culture.

  • a.
    Add poly-L-ornithine solution to the plates you plan to passage the cells, and then rock and shake the plates to be sure the entire surface is covered by liquid. Incubate the plates in the incubator at 37°C for 2 h. Remove the majority of the poly-L-ornithine solution from the plates and wash the plates 3 times with sterile Milli-Q H₂O.

    CRITICAL: From this point on, the plates must not be allowed to dry out.

  • b.
    Add laminin solution to the plates and incubate the plates in the incubator at 37°C for 2 h. Remove majority of the laminin solution and wash the plates 3 times with sterile PBS.
  • c.
    After the 7–14 days of culture, collect all primary spheres to a 15 mL conical tube without disturbing the attached cells; spin at 200 g for 5 min at room temperature.
  • d.
    Carefully remove the medium and add 750 μL of 0.05% trypsin/EDTA to the tube. Incubate the tube in the incubator for 3 min at 37°C. Filter the media with 20 μm filter. Keep conditioned medium!
  • e.
    Dissociate the spheres using a pre-wet 1 mL blue tip by pipetting up and down 20 times to digest the spheres within 2 min.
  • f.
    Incubate tube again in the water bath for 2 min at 37°C. Dissociate neurospheres again using a 200 μL pipette.
  • g.
    Add 750 μL of Solution C to the tube and triturate up and down 10 more times until no clumps are visible anymore.
  • h.
    Add 5 mL of AGM to each tube, mix by inverting the tubes a few times, and pellet the cells at 200 g for 5 min at room temperature. Remove the supernatant (medium) and collect it in a separate flask.
  • i.
    Centrifuge for 5 min at 1000 rpm at 4°C in a centrifuge.
  • j.
    Remove the supernatant and re-suspend the cells in 1 mL of AGM. Triturate with a 1 mL pipette.
  • k.
    Plate the neural progenitor cells into a coated 24-well plate and incubate the cells in a 5% CO₂ incubator at 37°C.

    CRITICAL: Avoid the formation of air bubbles. Excessive air bubbles will lower the cell viability and enhance the possibility of contamination.

Telomerase activity detection in neural progenitor cells by non-radioactive TRAP method

Timing: 2 h (for step 4)

Timing: 20 min (for step 5)

Timing: 2–3 h (for step 6)

Timing: 1–2 h (for step 7)

This section outlines a non-radioactive method for detecting telomerase activity in neural progenitor cells (NPCs) using the Telomeric Repeat Amplification Protocol (TRAP) assay, providing a sensitive analysis without the use of radioisotopes. Understanding telomerase activity in NPCs aids in elucidating their role in neurogenesis and aging-related diseases, offering insights into cellular aging mechanisms and potential therapeutic strategies.

• 4.
  Preparation of Telomerase extracts from mouse neural progenitor cells.

  CRITICAL: Prior to initiating the extraction process, it is crucial to establish a specialized laboratory setup and implement significant precautions to prevent PCR carry-over contamination and RNase contamination. This involves utilizing a clean bench top, nuclease-free water, and sterile laboratory consumables such as pipettes and tips.

  • a.
    Begin by harvesting 10⁵ 10⁶ neural progenitor cells into a sterile microcentrifuge tube that is free from DNase and RNase.
  • b.
    Pellet the cells by centrifugation in a tabletop centrifuge at 10,000 g for 5 min at room temperature (20°C–25°C). Carefully remove and discard the supernatant.

    Note: If samples need to be stored for future use, promptly freeze the cell pellet in liquid nitrogen and store it at −80°C until ready for lysis.

  • c.
    Resuspend the cell pellet in 200 μL ice-cold lysis buffer by gently pipetting up and down 3–5 times using a 200 μL tip.
  • d.
    Allow the cells to incubate on ice for 30 min, then centrifuge the homogenates at 10,000 g for 20 min at 4°C.

    Note: Ensure all subsequent steps are performed under cold conditions.

  • e.
    Transfer the resulting supernatant into a fresh tube and determine the protein concentration using the Bradford assay.

    Note: To prevent frequent freeze-thaw cycles, aliquot the supernatant into smaller volumes, snap-freeze it on dry ice, and store it at −80°C. The extract remains stable for at least 12 months at −80°C.

    CRITICAL: Exercise caution to avoid RNase contamination at every stage of the TRAP assay.

• 5.Protein estimation by Bradford assay.
  Bio-Rad Protein Assay was used to determine the protein concentration in the extracts from neural progenitor cells.

  • a.
    Prepare a series of BSA standard solutions with concentrations ranging from 0 to 1 mg/mL.
  • b.
    Add 1 mL of Bradford protein assay reagent to each well of a microplate.
  • c.
    Add 5 μL of each BSA standard solution to separate wells containing the Bradford reagent. Also, include a well with 5 μL of nuclease-free water.
  • d.
    Mix the contents thoroughly by vortexing or pipetting.
  • e.
    Incubate the microplate at room temperature for 5–10 min to allow for color development.
  • f.
    Measure the absorbance of each sample at 595 nm using a spectrophotometer.
  • g.
    Plot a standard curve of absorbance versus protein concentration using the absorbance values obtained from the BSA standard solutions.
  • h.
    Determine the protein concentration of the sample by interpolating its absorbance value from the standard curve.

    Note: Use fresh BSA standard solutions to prevent degradation and to ensure accuracy of the standard curve. Adjust the sample volume and dilution factor if necessary to ensure that the absorbance falls within the linear range of the standard curve.

    Note: The most effective protein concentration for PCR reactions in neural progenitor cells were identified to be within the range of 1 μg/ μL.

• 6.
  TRAP reaction.

  • a.
    Preparation of control samples:

    • i.
      Sample control: As telomerase is a heat-sensitive enzyme, every sample should be analyzed in two ways: one with heat treatment (serves as negative control, heat inactivated at 85°C for 10 min) and without heat treatment.
    • ii.
      Telomerase-positive control: The HeLa cell line is chosen as a positive control to measure telomerase activity. The cell extract is resuspended in lysis buffer, aliquoted into tubes, and stored at −80°C.
    • iii.
      PCR Amplification control: This internal control band assess the efficiency of amplification in a PCR reaction and monitors any inhibition of PCR. It generates a 36 bp band in every lane except no Taq control.
    • iv.
      PCR contamination control: This control contains lysis buffer and nuclease-free water as sample, serving as a substitute for the extract. This will only amplify a 36 bp internal control band.
    • v.
      No Taq control: No PCR products or internal control band will be visible in this lane.
  • b.
    The TRAP comprises two steps:

    CRITICAL: Perform all the pipetting steps outlined in the protocol using filter tips to minimize the risk of contamination. Maintain all PCR reagents and samples on ice throughout the procedure.

  • c.
    Primer extension:

    • i.
      Prepare the telomerase reaction mixture by combining the following components in a microcentrifuge tube: 2 μL of TRAP mix (10×), 0.5 μL of TS primer, 0.5 μL of dNTPs (10 mM), and 16 μL of nuclease-free water (Table 1). If handling multiple samples, multiply the indicated volumes by the number of samples and add 1.
    • ii.
      Next, dispense 19 μL of the reaction mixture into each designated reaction tube. Then, add 1 μL of the diluted protein extract (1 μg) to each tube, achieving a final volume of 20 μL.
    • iii.
      Proceed to incubate the reaction tubes in a PCR machine at 30°C for 30 min
  • d.
    PCR amplification:

    • i.
      Prepare PCR master mix by adding the following components to a micro-centrifuge tube: 2.5 μL Titanium Taq PCR Buffer (10×), 0.5 μL 1× TRAP primer mix, 2.6 μL nuclease-free water, 0.2 μL Titanium Taq DNA Polymerase (50×) and 0.2 μL BSA (20 μg/mL) (Table 1). For multiple samples, increase the previously mentioned volumes by the number of samples plus 1.
    • ii.
      Dispense 6 μL of the PCR mix into each tube, then add the entire volume of the telomerase reaction (20 μL, prepared in Step I) to each tube, resulting in a final volume of 26 μL.
    • iii.
      Insert the PCR tubes into a thermo cycler and run the following program: 95°C–2 min, ∗93.4°C–30 s, ∗53.2°C–30 s, ∗68°C–45 s, 68°C–5 min, ∗ = 34 cycles.

      Note: Following PCR, samples can be stored at 4°C for a maximum of 2 days or at −20°C for an extended duration until they are analyzed on an acrylamide gel.

• 7.
  Detection of Telomerase activity.

  • a.
    Prepare a 10% nondenaturing acrylamide gel in 0.5× TBE solution. The gel recipe involves combining the following components: Distilled Water – 8.4 mL, 19:1 acrylamide:bis-acrylamide – 3 mL, TBE (10×) – 600 μL, 10% APS – 100 μL, and TEMED – 15 μL.

    CRITICAL: Proper gel preparation is crucial to ensure that TRAP ladder bands are well-separated and run effectively in each lane. Add freshly prepared APS, and perform thorough mixing after adding all components.

  • b.
    Pour the gel into a vertical mini-gel apparatus (12 × 16 × 18 cm). Once the gel solidifies at room temperature, allow it to cool for 30 min at 4°C before usage.
  • c.
    Load each lane with 10 μL of TRAP sample (prepared from 20 μL TRAP PCR products and 5 μL 6× gel-loading dye), then run the gel at 110 V for 2 h

    Note: Prevent excessive sample loading onto the gel, as it could result in band smearing.

  • d.
    Prepare a 3× working solution of nucleic acid stain by adding 15 μL of 10,000× nucleic-acid stain stock solution to 50 mL of Distilled Water containing 0.1 M NaCl. Stain the gel for 20 min with gentle shaking (45–50 RPM).
  • e.
    Finally, utilize a UV transilluminator emitting light at a wavelength of 302 nm to visualize the TRAP ladder DNA bands.
• 8.
  Real-time Quantitative TRAP assay.

  • a.
    For the real-time Quantitative TRAP assay (Q-TRAP), follow the same protocol for cell extraction as in 4.
  • b.
    The PCR reaction mixture can be prepared by adding 1× PowerUp SYBR Green Master Mix, 100 ng TS primer per sample, 100 ng ACX primer per sample, 10 mM EGTA and water for the appropriate number of samples (n+1), keeping final volume of the reaction mix to 25 μL per sample (Table 2).
  • c.
    Incubate the plate for 30 min at 30°C in the dark for extension of the substrate by telomerase.
  • d.
    Insert the plates into Roche LC 480 PCR machine and run the following program: 95°C for 10 min (to activate the polymerase); 40 cycles at 95°C for 30 s and at 60°C for 90 s.

Table 1.

TRAP assay

Component	Volume, 1×
Step I
Nuclease-free H2O	16 μL
10× TRAP buffer	2.0 μL
50× dNTP mix	0.5 μL
TS primer (100 ng/mL)	0.5 μL
Step II
Nuclease-free H2O	2.6 μL
10× Titanium Taq PCR Buffer	2.5 μL
50× TRAP primer mix	0.5 μL
BSA (50 mg/mL)	0.2 μL
50× Titanium Taq DNA polymerase	0.2 μL

Table 2.

Q-TRAP assay

Component	Volume, 1×
Nuclease-free H2O	6
PowerUp SYBR Green PCR master mix	12.5
EGTA (10 mM)	2.5
TS primer (100 ng/μL)	1
ACX primer (100 ng/μL)	1
Total	23

Expected outcomes

This method is designed to efficiently isolate neural progenitor cells from the hippocampus of mice aged 30–40 weeks. Under optimal conditions outlined here, neurospheres are anticipated to develop within two weeks following isolation. Failure to observe sphere formation within the expected time frame may indicate unsuccessful isolation, and prolonging the culture time is unlikely to yield positive results based on our experience.

Neural progenitor cells isolated from hippocampus express NPC markers such as Nestin and Sox2 (Figures 1A and 1B for neurospheres; Figure 1C for monolayer culture), and demonstrate proliferative capacity, as evidenced by BrdU incorporation (Figure 1D). NPCs extracted from the hippocampus can be induced to differentiate into neurons using specific differentiation media. Immunocytochemistry, as depicted in Figure 1E, confirms the lineage of differentiated cells, with neurons identified by Tuj1 staining.

Figure 1.

Isolation of NPCs from the hippocampus of the aged mice and subsequent analysis of telomerase activity in NPCs

(A and B) Representative image of neurospheres from hippocampus that appeared at 7 days after initial plating (passage 0) as shown by (A) Phase contrast microscope, (B) Immunostaining - Sox2 (red) and Nestin (green) neuronal markers. Nucleus is stained by DAPI (blue).

(C) Representative image of a monolayer adherent NPC culture from hippocampus marked by Sox2 (red) and Nestin (green). Nucleus is stained by DAPI (blue).

(D) Representative image showing incorporation of BrdU in NPCs (green) and nuclei counterstained by DAPI (blue).

(E) Immunostaining of differentiated NPCs highlighted in neuronal differentiation marker Tuj1 (red) and nuclei counterstained by DAPI (blue).

Image scale bars = 10 μm.

(F) Mouse NPCs from aged hippocampus are shown to be positive for telomerase activity, as evidenced by the 6-bp incremental TRAP ladder. HeLa cell extract served as positive control. The heat-inactivated (HI) NPC cell lysates, lysis buffer only, water only, and no taq control served as negative controls (NC). Each TRAP reaction includes a 36-bp internal standard control (IC). Serial dilutions of NPCs result in a decreased intensity of the telomerase products TRAP ladder.

(G) The human HeLa cell line extract are shown to be positive for telomerase activity, as evidenced by the 6-bp incremental TRAP ladder and serial dilutions results in a decreased intensity of the telomerase products TRAP ladder. Lysis buffer, water and heat-inactivated (HI) cell lysates served as negative controls (NC). Each TRAP reaction includes a 36-bp internal standard control (IC).

(H) Determination of the standard curve and linear relationship for Q-TRAP. The Ct values (±s.d.) of the standard control are plotted against log[protein] to calculate the linear equation. The Y-intercept and the slope values from the equation are used to quantify the RTA of unknown samples.

(I) Samples were quantified as described in the protocol and plotted as RTA ± s.d. For Q-TRAP quantification, the RTA for an unknown sample was calculated based on the following standard curve and equation obtained for the same Q-TRAP assay: y = −2.9763x + 33.285; RTA = 10^{[(Ct sample−Yint)/slope]}.

Under recommended conditions, hippocampus-derived NPCs can be passaged for extended periods (>20 passages) without losing their NPC characteristics. However, prolonged passaging may increase chromosome abnormalities and diminish the ability to differentiate into neurons. NPCs obtained using this method from adult mice can be utilized for gene expression and biochemical analyses.

As telomerase activity diminishes with age, this protocol offers a means to identify telomerase products in NPCs of an older mice aged 30–40 weeks. Protein extracts were obtained from neural progenitor cells isolated from the hippocampus of mice within this age range. Telomerase activity was then assessed in 1 μg of protein extract using TRAP assay. Our method of detecting telomerase activity in NPCs demonstrates improved resolution of telomerase DNA products in the adult mice, as evidenced by the length and intensity of the DNA ladder observed (Figure 1F).

The Telomerase activity in NPCs were detected in a two-step process. In the first step during the TRAP assay, different amounts of hexameric repeats get added to it within the first 30 min of incubation with the TS primer. In the next step, when we amplify these products later, we see a ladder pattern on the gel, with each ladder representing an increment of 6 base pairs in DNA length (Figure 1F). This ladder gives us a measure of how much telomerase activity is present per unit of protein used in the assay. Additionally, we used an internal control along with the TRAP primer mix. We observed a 36-base pair band on the acrylamide gel which serves as a control for PCR amplification efficiency and can be used for semi quantitative analysis (Figure 1F). We additionally validated our method using the HeLa human cell line (immortalized cervical cancer cells and is known for its elevated telomerase activity), employed as a positive control (Figure 1F) and also performed a dilution series with similar protein amounts (in μg) per cell equivalent (Figure 1G).

The quantitative detection of telomerase activity in real-time involves measuring sample concentrations using a standard curve in a PCR machine. In our protocol, we normalized for protein amounts (in μg) per cell equivalent. By repeating the serial dilution series for neural progenitor cells (NPCs), as depicted in Figure 1F and utilizing the relative standard curve method for real-time data analysis, we plotted the Ct values of the samples against log[protein] to derive the linear equation (Figure 1H). The coefficient of determination (R2) should exceed 0.90. The Y-intercept and slope values obtained from this equation were then utilized to quantify the relative telomerase activity (RTA) of NPCs and HeLa cell extracts (Figure 1I, RTA of unknown sample = 10 ^{[(Ct sample−Yint)/slope]}). Employing the relative standard curve method as outlined above enables comparison of the telomerase activity of one sample with another sample performed in the same experiment, which is the standard approach for analyzing real-time PCR products.

Limitations

The study focuses on isolating neural progenitor cells (NPCs) from the hippocampus of mice aged 30–40 weeks. Therefore, the applicability of the method to much older mice, as well as other species, remains unexplored. The effectiveness of the protocol in isolating NPCs from different age groups or species needs further investigation.

Although NPCs can be passaged for extended periods without losing their characteristics, prolonged passaging may lead to increased chromosome abnormalities and diminished differentiation capacity. This limitation highlights the need for caution when using extensively passaged NPCs for downstream analyses, as their properties may be altered.

While the study presents a method for detecting telomerase activity in NPCs, it focuses on a specific age range (30–40 weeks) of mice. The applicability of the method to NPCs from mice of older ages or other organisms remains to be determined. Telomerase activity can vary with age, with higher activity typically observed in younger individuals and lower activity in older individuals. Therefore, the method optimized for NPCs from mice aged 30–40 weeks may not be directly applicable to older age groups where telomerase activity levels may differ significantly. Furthermore, telomerase regulation may differ in other organisms, including humans or other animal models. For example, the sensitivity and specificity of the assay, as well as potential interference from other cellular factors, may differ between samples from different age groups or species. Therefore, optimization of the assay conditions and careful interpretation of the results may be required to account for potential age- or species-related differences in telomerase regulation.

Despite precautions outlined for minimizing PCR carry-over contamination, such as using clean laboratory conditions and nuclease-free reagents, the risk of contamination during the TRAP assay cannot be completely eliminated. Contamination events may lead to false-positive results and undermine the validity of telomerase activity measurements.

While the protocol provides detailed instructions for TRAP assay execution and gel visualization, interpreting the results requires careful consideration of various factors, including band intensity, ladder pattern, and control samples, and distinguishing specific bands from background noise or artifacts, potentially leading to misinterpretation of telomerase activity levels in isolated NPCs.

Troubleshooting

Problem 1: Low cell yield

The problem of low cell yield arises due to various factors such as loss of brain tissue during dissection, insufficient mechanical dissociation, and cell adherence to the Pasteur pipette.

• •Loss of Brain Tissue During Dissection: This can occur if the dissection skills are not optimal, leading to the unintentional collection of surrounding tissues along with the desired hippocampus tissue.
• •Insufficient Mechanical Dissociation: Incomplete breakdown of tissue into single cells may result from insufficient mechanical dissociation, where tissue clumps remain despite the dissociation process.
• •Cell Adherence to the Pasteur Pipette: Cells sticking to the surface of the Pasteur pipette during trituration can reduce the yield of isolated cells, as they fail to be released into the dissociation media.

Potential solution

• •Improve Dissection Skills: Enhance dissection skills to ensure only the hippocampus tissue is collected, minimizing the loss of valuable material. Optimal dissection techniques prevent the inadvertent inclusion of surrounding tissues, resulting in a higher yield of desired cells.
• •Extend Digestion Time: Extend the digestion time in Dissociation media up to 30 min to ensure complete tissue breakdown. Prolonged digestion allows for thorough dissociation of tissue clumps into single cells, maximizing the yield of isolated cells for subsequent experiments.
• •Pre-wet the Pasteur Pipette: Pre-wet the Pasteur pipette before trituration to prevent cell adherence and enhance the recovery of isolated cells. Wetting the pipette surface reduces the likelihood of cells sticking to it during the trituration process, facilitating their release into the dissociation media and improving cell yield.

Problem 2: Excess debris in culture

Excess debris in the culture can result from the presence of excessive striatal tissue, dead cells, and myelin contaminants.

• •Presence of excessive striatal tissue, dead cells, and myelin contaminants may occur if the dissection precision is not optimal or if the tissue is not properly inspected before trituration.

Potential solution

• •Improve Dissection Precision: To address the presence of excess debris in the culture, improve dissection precision by aiming for thinner hippocampus tissue cuts. Thinner cuts reduce the likelihood of including surrounding tissues such as striatal tissue, minimizing contamination in the culture.
• •Inspect Culture Under the Microscope: After trituration, inspect the culture under the microscope to ensure the absence of aggregates and debris. A clean culture environment devoid of debris and contaminants indicates successful removal of excess tissue and ensures a healthy culture for subsequent experiments.

Problem 3: Few or no visible TRAP ladder bands

The absence or low visibility of TRAP ladder bands can result from factors such as low starting material, low protein concentration, and loss of samples during processing.

• •Low starting material may occur if the cell count is insufficient for visualization and quantification of TRAP ladder bands.
• •Low protein concentration may result from errors in the setup or preparation of reagents.
• •Loss of samples during processing can happen due to technical errors or mishandling.

Potential solution

• •Ensure Sufficient Cell Count: Ensure a minimum of 100,000 cells are used for the TRAP assay to improve the visibility and quantification of ladder bands on the gel. Increasing the starting material enhances the signal-to-noise ratio and improves the reliability of the assay.
• •Review Reagent Preparation: Review the components added during setup and adjust concentrations if necessary to ensure optimal protein concentration in the samples. Proper reagent preparation is critical for obtaining accurate and reproducible results in the TRAP assay.
• •Optimize PCR Cycling Conditions: Optimize PCR cycling conditions to improve amplification efficiency and reduce background noise. Adjust parameters such as denaturation temperature, annealing temperature, and extension time to optimize the PCR reaction for robust amplification of telomeric DNA fragments.

Problem 4: TRAP products visible in negative control lanes

Presence of TRAP products in negative control lanes indicates potential contamination with RNase/DNase or PCR carry-over contamination.

• •RNase/DNase contamination may occur due to inadequate laboratory hygiene practices.
• •PCR carry-over contamination can result from the transfer of PCR products or reagents between samples during handling.

Potential solution

• •Sterilize the Laboratory Bench and Equipment: Regularly sterilize the laboratory bench and equipment to minimize the risk of contamination. Use disinfectants to clean surfaces thoroughly, and maintain good laboratory hygiene practices.
• •Use RNase Inhibitor and Fresh Reagents: Add RNase inhibitor to the 1× Lysis Buffer and use fresh reagents to prevent enzymatic degradation of RNA. RNase inhibitors effectively inhibit the activity of RNases, ensuring the integrity of RNA samples during processing.
• •Avoid Overloading Lanes with Protein: To prevent contamination and minimize the risk of false-positive results, avoid overloading lanes with protein during gel electrophoresis. Carefully handle samples to prevent cross-contamination between lanes.

Problem 5: Bands in heat-inactivated cell extract

Presence of bands in heat-inactivated cell extract lanes suggests inadequate heat inactivation or PCR carry-over contamination.

• •Inadequate heat inactivation occurs when the samples are not heated for a sufficient duration or temperature to denature telomerase activity.
• •PCR carry-over contamination may occur if precautions are not taken to prevent contamination during handling and processing.

Potential solution

• •Heat Samples at 85°C for at Least 10 min: To ensure complete denaturation of telomerase activity, heat the samples at 85°C for at least 10 min. This extended heating duration ensures thorough denaturation of telomerase, minimizing the risk of residual activity and the appearance of bands on the gel.
• •PCR Carry-over Contamination: If PCR carry-over contamination is suspected, refer to the solutions outlined in the troubleshooting 4 for PCR contamination. These solutions may include measures such as using fresh reagents, avoiding contamination during sample handling, and implementing rigorous decontamination procedures in the laboratory.

Problem 6: Extra bands between 36 bp internal control and TRAP ladder bands

Presence of extra bands between the 36 bp internal control and TRAP ladder bands indicates potential high telomerase activity in sample extracts.

• •High telomerase activity may result from an excessive concentration of template DNA or suboptimal PCR conditions.

Potential solution

• •Dilute Sample Extracts: To address high telomerase activity, it is recommended to dilute the sample extracts. Dilution reduces the concentration of template DNA in the reaction, thereby decreasing the likelihood of non-specific amplification and the appearance of extra bands.
• •Optimize PCR Conditions: Optimize PCR conditions to minimize non-specific amplification and improve specificity. This includes determining the denaturation and annealing temperatures suitable for each PCR machine and adjusting reagent concentrations as needed. Optimization ensures efficient amplification of the target DNA fragments while minimizing the generation of unwanted products.
• •Determine Denaturation and Annealing Temperatures: Determine the optimal denaturation and annealing temperatures for each PCR machine used in the experiment. Variations in PCR machine performance may require adjusting the temperature settings to achieve optimal amplification efficiency and specificity.
• •Adjust Reagent Concentrations: Evaluate and adjust the concentrations of PCR reagents such as primers, dNTPs (deoxyribonucleotide triphosphates), and Taq Polymerase to optimize PCR conditions. The appropriate reagent concentrations ensure robust amplification of target DNA fragments while reducing non-specific amplification.

Problem 7: Smearing of TRAP ladder bands

Smearing of TRAP ladder bands suggests potential protein overload or technical issues during gel preparation.

• •Protein overload occurs when an excessive amount of protein is loaded onto the gel, leading to distorted band patterns.

Potential solution

• •Dilute the Starting Material: To address protein overload, it is recommended to dilute the starting material. By reducing the protein concentration, you can alleviate the overcrowding of the gel lanes, which can lead to clearer and more defined bands.
• •Avoid Overloading Lanes with Protein: Additionally, it’s crucial to ensure that gel lanes are not overloaded with protein. Proper loading of samples helps prevent overcrowding and allows DNA fragments to migrate more effectively, resulting in better band resolution.
• •Incorporate Hot Start Taq Polymerase: Hot start Taq Polymerase is an enzyme that remains inactive at lower temperatures, preventing non-specific amplification of DNA during the initial stages of PCR setup. By incorporating Hot start Taq Polymerase into the reaction, you can minimize the likelihood of non-specific amplification, which could contribute to smearing of bands on the gel.

Problem 8: Disoriented ladder bands and lane

Disoriented ladder bands and lanes indicate potential issues with gel preparation, including errors in mixing or incorrect concentration of reagents.

• •Errors in gel preparation may result from inaccuracies in adding components or improper mixing of solutions, leading to distorted band patterns.

Potential solution

• •Review the gel preparation method and recipe to ensure all components are added accurately and thoroughly mixed.
• •Ensure thorough mixing of components and utilize freshly prepared APS for polymerization.
• •Employ the correct concentration of APS to ensure proper gel formation.

Problem 9: Low Ct value

A low Ct value suggests unoptimized PCR conditions, background noise, or contamination.

• •Unoptimized PCR Conditions and Background Noise: Suboptimal PCR conditions such as incorrect primer concentrations, annealing temperature, or extension time can lead to low Ct values and increased background noise in the PCR reaction.
• •Contamination: Presence of contaminants in the PCR reaction mix, such as nucleic acids or enzymes from previous reactions, can lead to lower Ct values.

Potential solution

• •Optimize PCR Conditions: Adjust primer concentrations, annealing temperature, and extension time to optimize PCR conditions for improved specificity and efficiency. Perform gradient PCR or optimization experiments to determine the optimal parameters.
• •Check Taq Polymerase Activity: Ensure that the Taq Polymerase enzyme is still active by performing a positive control PCR reaction with known template DNA. If the positive control does not yield the expected results, consider replacing the Taq Polymerase with a fresh stock.
• •Practice Sterile Conditions: Minimize the risk of contamination by working in a clean and sterile environment. Use filtered pipette tips, sterile tubes, and decontaminate work surfaces regularly. Perform negative control reactions alongside experimental samples to monitor for contamination.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, [D.K] (diji.kuriakose1@monash.edu).

Technical contact

Questions about the technical specifics of performing the protocol should be directed to and will be answered by the technical contact, [D.K] (diji.kuriakose1@monash.edu).

Materials availability

The requests for materials and reagents used in this protocol can be directed towards [D.K] (diji.kuriakose1@monash.edu).

This study did not generate new unique reagents.

Data and code availability

This study did not generate/analyze datasets/code.