Protocol for metabolite extraction and sample preparation from primary astrocytes for mass spectrometry-based metabolomic analysis

Summary

Astrocytes play essential roles in supporting neuronal function, particularly through the regulation of brain energy metabolism. In response to physiological and pathological stimuli, astrocytes dynamically adjust their metabolic pathways and energy output. Here, we present a protocol for metabolite extraction and sample preparation from primary astrocytes for mass spectrometry analysis. We describe steps for integrating astrocyte culture and liquid chromatography-mass spectrometry (LC-MS) metabolite analysis to enable reproducible profiling of astrocytic energy metabolism under different experimental conditions.

For complete details on the use and execution of this protocol, please refer to Chang et al.¹

Graphical abstract

Highlights

• •Primary astrocyte culture with freeze-thaw preservation and cytokine activation
• •Step-by-step PCA-based metabolite extraction and protein precipitation
• •Guidance on metabolite quantification using fenclonine and protein normalization

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

Astrocytes play essential roles in supporting neuronal function, particularly through the regulation of brain energy metabolism. In response to physiological and pathological stimuli, astrocytes dynamically adjust their metabolic pathways and energy output. Here, we present a protocol for metabolite extraction and sample preparation from primary astrocytes for mass spectrometry analysis. We describe steps for integrating astrocyte culture and liquid chromatography-mass spectrometry (LC-MS) metabolite analysis to enable reproducible profiling of astrocytic energy metabolism under different experimental conditions.

Before you begin

Astrocytes are critical glial cells that provide essential metabolic and trophic support to neurons,² maintaining the energy homeostasis required for normal brain function.³ Although astrocyte behavior can differ between in vitro and in vivo contexts, primary astrocyte cultures offer a simplified and well-controlled model system⁴ for studying their regulatory mechanisms under physiological and pathological conditions.⁵

In this protocol, we describe the isolation and culture of primary cortical astrocytes, the induction of inflammatory responses using pro-inflammatory cytokines, and the preparation of samples using a perchloric acid (PCA)-based extraction method for metabolomic profiling by UPLC-MS^6,7,8,9 (Figure 1A). Details regarding the setup, tuning, and calibration of the UPLC-MS instrument are not included here. Instead, this protocol focuses on sample preparation, quantification of targeted metabolites using Fenclonine as an internal standard, and normalization to total protein content.

Figure 1.

Workflow for culturing primary astrocytes and assessing purity and cytokine-induced activation

(A) Schematic overview of the experimental workflow for isolating and culturing primary astrocytes from P0–P2 mouse cortices.

(B) Instruments and setup used for mouse brain dissection.

(C) Step-by-step demonstration of handling and dissection of P1 mice: holding the head (C1), removing the skin (C2), removing the skull (C3), and exposing the brain tissue (C4).

(D) The skin and skull of the mouse are cut and removed using precision surgical scissors.

(E) Images showing removal of the meninges and collection of target brain regions (cortex, hippocampus, and striatum) from the P1 mouse brain.

(F) Representative images showing the washing and trituration procedures of brain tissue.

(G) Images illustrating the pipette tip configurations used for trituration and homogenization of brain tissue.

(H) Representative bright-field images showing the morphology of cultured primary astrocytes. Black arrows indicate suspended cells floating above the adherent astrocyte layer.

(I) Immunostaining of GFAP in cultured primary astrocytes.

(J) Transcript levels of cell type–enriched genes, including astrocytic (Glul, Slc1a3, Aqp4), microglial (Aif1), neuronal (Tubb3), oligodendrocyte (Mbp), and NG2 glial (Cspg), measured by qPCR. Ct values were normalized to ribosomal 18S (r18S).

(K) Expression levels of reactive astrocyte genes under basal and cytokine-treated conditions. Data are represented as mean ± SEM and analyzed using Student’s t test. ∗∗∗∗, p < 0.0001.

All animal procedures described in this protocol were approved by the Academia Sinica Institutional Animal Care and Use Committee (IACUC), Taiwan (Protocol No. 24-07-2217), and were conducted in accordance with institutional guidelines for the care and use of laboratory animals. Investigators intending to implement this protocol should review their local institutional requirements and obtain ethical approval as appropriate.

Innovation

Although protocols for primary astrocyte culture and UPLC-MS metabolite analysis have been described separately, a unified workflow specifically optimized for small-molecule energy metabolite profiling using primary astrocytes is currently lacking. This protocol integrates primary astrocyte culture, cytokine-induced activation, and PCA-based metabolite extraction into a single reproducible workflow compatible with UPLC-MS analysis.

By linking astrocyte culture procedures with metabolomic sample preparation and quantitative normalization strategies, this protocol enables reliable assessment of astrocytic metabolic states under defined experimental conditions. The protocol also incorporates practical guidance for metabolomic sample preparation, including procedures for protein precipitation, metabolite normalization using an internal standard and total protein content, and strategies to minimize salt contamination during sample processing.

Importantly, this integrated approach provides a practical and reproducible framework for investigating astrocyte metabolic adaptations and facilitates studies that examine how astrocytes regulate energy metabolism during physiological responses and inflammatory activation.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Chemicals, peptides, and recombinant proteins
DMEM	Hyclone	Cat# SH30003.02
Poly-L-lysine	Sigma-Aldrich	Cat# P1274
L-glutamine	Gibco	Cat# 25030-081
Penicillin/Streptomycin	Gibco	Cat# 15140-122
DMSO	Sigma-Aldrich	Cat# D5879-500mL
Mouse TNFα	Sigma-Aldrich	Cat# T7539-50UG
Mouse IL1α	Sigma-Aldrich	Cat# I5396-25UG
Trypsin-EDTA	Gibco	Cat# 15400-054
Perchloric acid (PCA)	Sigma-Aldrich	Cat# 311413
Potassium carbonate (K2CO3)	Sigma-Aldrich	Cat# 209619
Acetonitrile (ACN)	Supelco	Cat# 1.00029.2500
MeOH	Macron Chemicals	Cat# 3016-22
Acetone	Sigma-Aldrich	Cat# 179973
DTT	Sigma-Aldrich	Cat# D0632
urea	PROTECH	Cat# CH35940
thiourea	Sigma-Aldrich	Cat# T7875
CHAPS	Sigma-Aldrich	Cat# C3023
Fenclonine	Sigma-Aldrich	Cat# C6506-25G
Critical commercial assays
PierceTM BCA Assay Kit	Thermo Fisher	Cat# 23225
Other
MX-160 High Speed Refrigerated microcentrifuge	TOMY	MX-160
SpeedVac Vacuum Concentrator	Savant	Cat# 75040 Dryer 8
Bioruptor Plus sonication device	Diagenode	Cat# B01020014
ACQUITY UPLC BEH Amide column (2.1 × 100 mm, 1.7 μm)	Waters	SKU# 176001908
Agilent 6545 XT Q-TOF LC/MS	Agilent	G6549A
2 ml Screw Top Vials	Agilent	Part# 5182-0715
2 ml Screw Top Tops	Agilent	Part# 5185-5820
250 μL Vial inserts	Agilent	Part# 51904073
1250 μL Tip	starmoonbio	Cat# SMB-T1250RL
200 μL Tip	starmoonbio	Cat# SMB-T200RL
10 μL Tip	starmoonbio	Cat# SMB-T10RL
1.5 mL Microcentrifuge Tube	starmoonbio	Cat# M1540-C
15 mL High Clarity Centrifuge Tube	Falcon	Cat# 352096

Materials and equipment

Poly-L-lysine preparation

Prepare the stock solution at 0.5 mg/mL in ddH₂O. Sterilize by filtration using a 0.22 μm filter unit and store at 4°C for up to 6 months.

Before use, dilute the poly-L-lysine stock 1:5 in ddH₂O to a final concentration of 0.1 mg/ml. Store the working solution at 4°C and use within one month.

Astrocyte culture medium

Reagent	Final concentration	Amount
DMEM	N/A	440 mL
L-glutamine	1%	5 mL
Penicillin/Streptomycin	1 %	5 mL
FBS	10 %	50 mL
Total	N/A	500 mL

Store at 4°C and pre-warm before use.

Note: This protocol uses primary astrocytes cultured in medium containing 10% FBS. Because emerging studies indicate that serum can influence astrocyte reactivity and potentially confound experimental interpretation,^10,11 we acknowledge the increasing adoption of serum-free culture systems. These serum-free conditions are not included in this protocol and should be independently optimized by the user.

Cytokines mixture to induce reactive astrocytes

Reagent	Final concentration	Amount
Astrocyte culture medium	N/A	5 ml
TNFα (stock: 100 μg/ml)	30 ng/mL	1.5 μL
IL-1α (stock: 10 μg/ml)	3 ng/mL	1.5 μL
Total	N/A	5 ml

After dissolving TNF-α and IL-1α, aliquot the solution (10–20 μL) into several vials and store them at −20°C to preserve activity and minimize freeze–thaw cycles.

0.6 N perchloric acid (PCA) for metabolites extraction

Reagent	Final concentration	Amount
Fenclonine (stock: 10 ppm)	2.3 ppm (before K2CO3 neutralization) 2 ppm (after K2CO3 neutralization)	184 μl
PCA (stock: 60%)	0.6 N	264 μl
ddH2O	N/A	3552 μl
Total	–	4 ml

Prepare the solution fresh on the day of the experiment and maintain it on ice until use.

Note: Do not use sterilized ddH₂O to prepare the solution, as the sterilization process can release small molecules that may interfere with the analysis.

CRITICAL: Perchloric acid (PCA) is highly corrosive and may generate explosive perchlorate residues. Perform all procedures in a suitable fume hood, and wear appropriate personal protective equipment, including a lab coat and acid-resistant gloves. Use only glass or compatible plastic lab ware. Always add acid to water, never water to acid.

ACN/MeOH/ddH₂O solvent for UPLC

Reagent	Final concentration	Amount
ACN	40 %	400 μL
MeOH	40 %	400 μL
ddH2O	20 %	200 μL
Total	N/A	1 ml

Prepare the solution fresh on the day of the experiment. Use filtered ddH₂O and avoid using sterilized water. Keep the solution in 4°C until use.

20 mM DTT in acetone for washing protein pellets

Reagent	Final concentration	Amount
DTT (1M)	20 mM	1 mL
Acetone	N/A	49 mL
Total	N/A	50 mL

Store the prepared solution at 4°C until use.

2-D sample buffer to dissolve protein pellets for quantification

Reagent	Final concentration	Amount
Urea	6 M	9.1 g
Thiourea	1.5 M	2.85 g
CHAPS	3 %	0.75 g
DTT	60 mM DTT	231.3 mg
ddH2O	N/A	Up to 25 mL
Total	N/A	25 mL

Warm the solution in a 37°C water bath until fully dissolved, then store it at 4°C for longer-term use.

CRITICAL: Thiourea is a toxic reagent. Carefully review the SDS and use appropriate personal protective equipment when handling it.

Step-by-step method details

Primary astrocyte harvest from postnatal mice

Timing: 2–3 h

This section describes the isolation of brain tissue from postnatal mice, dissection of the target brain region, and mechanical dissociation of the tissue for primary astrocyte culture. These steps generate the initial mixed glial culture from which astrocytes are expanded for subsequent experiments.

Flask coating

• 1.Add 10 mL poly-L-lysine (0.1 mg/mL) to a T75 flask.
• 2.Incubate for 1 hour at room temperature (20°C −25°C) in a laminar-flow hood.
• 3.Remove the poly-L-lysine solution and aspirate excess liquid using sterile suction tips.
• 4.Wash the flask twice with sterile PBS or ddH₂O.

Note: If the flask will be stored for later use, wash it with ddH₂O and allow it to dry completely in the laminar-flow hood. Avoid washing with PBS to prevent salt precipitation.

• 5.Add 10 mL HBSS to the flask and keep it until cell plating.

Mouse brain collection, dissection, and homogenization

• 6.Place 4 ml ice-cold HBSS into a 3.5-cm dish and 10 ml ice-cold HBSS in a 15-mL tube on ice. (Figure 1B)
• 7.Spray neonatal mice (P0–P2) thoroughly with 70% ethanol for surface sterilization.
• 8.
  Brain isolation from P1 mice (Figure 1C).

  • a.
    Hold the body of the animal firmly with sterile tissue paper (Figure 1C1) to secure the head position and control cuts without compressing the brain.
  • b.
    Using fine surgical scissors (Figure 1D), make a posterior incision in the skin around the occipital region near the foramen magnum. Extend the incision rostrally along the dorsal midline toward the nasal region, as indicated by the dashed lines in Figure 1C2.
  • c.
    Gently retract and remove the skin using forceps to fully expose the skull surface.
  • d.
    Using fine surgical scissors, create a midline incision along the sagittal suture of the skull, beginning at the level of the foramen magnum and extending rostrally toward the frontal bone (Figure 1C3).
  • e.
    Carefully separate the two skull plates laterally using fine forceps to expose the brain surface, taking care not to damage the cortex or meninges, as neonatal brain tissue is particularly fragile.
  • f.
    Once the skull plates are peeled back (Figure 1C4), gently lift the brain from the cranial cavity and immediately transfer it into a 3.5-cm dish containing ice-cold HBSS.
• 9.Under a dissecting microscope, carefully remove the meninges and any remaining blood-colored tissue (Figures 1E1–1E3)
• 10.Isolate the target brain region(s) according to your experimental design (Figures 1E4–1E6).
• 11.Transfer the dissected tissue into a 15-mL tube containing 10 mL HBSS and wash the tissue 3 times with ice-cold HBSS (Figure 1F1).

Note: Allow the tissue to float in the center of the HBSS and wait for it to settle to the bottom of the tube before removing the wash solution. Discard the HBSS by gently inverting the tube, taking care not to lose the tissue, as it may slide out.

• 12.Completely remove the ice-cold HBSS (Figure 1F2).
• 13.Replace the HBSS with pre-warmed astrocyte culture medium before plating the cells into the T-75 flask.

Note: Keep the mouth/neck of the flask dry and clean to avoid contamination.

• 14.
  Add 1-mL astrocyte culture medium to each 15-mL tube for trituration (Figure 1F3).

  • a.
    Triturate the tissue 10 times using a 1-mL pipette (Figure 1G1) to mechanically dissociate the brain tissue into smaller pieces.
  • b.
    Allow the tube to stand undisturbed for 1 min to permit larger tissue fragments to settle at the bottom (Figure 1F4).
  • c.
    Triturate the supernatant 2–5 times using a P200 tip mounted on a P1000 pipette tip (configured as shown in Figure 1G2) until a uniform cell suspension is obtained (Figure 1F5).

Note: This step is critical for astrocyte viability and proliferation. Optimize the number of trituration cycles as needed, and avoid applying excessive force or generating bubbles during the process.

• 15.Transfer the cell-containing medium into a T75 flask preloaded with 10 mL DMEM.
• 16.Incubate at 37°C, 5% CO₂, in a humidified incubator for 24 h.
• 17.Replace the medium with 15 mL fresh astrocyte culture medium to remove dead cells one day after plating.
• 18.Maintain culture by changing 50% of the medium twice per week.

Astrocyte harvesting, storage, thawing, and plating

This section describes how to enrich, harvest, cryopreserve, thaw, and re-plate primary astrocytes for downstream applications. These procedures enable long-term storage of astrocyte cultures while maintaining sufficient viability and purity for experimental use.

Astrocyte harvesting and storage in liquid nitrogen

Timing: 1 h

• 19.At DIV10-14 (10–14 days in vitro), gently tap the side of the culture flask 10–20 times to mechanically detach loosely attached microglia from the astrocyte monolayer.

Note: When maintained under optimal conditions, the cells should reach > 95% confluence and establish a consistent monolayer.

Note: Microglia typically emerge between DIV10–14. When this occurs, you will see suspended cells floating above the adherent astrocyte layer¹² (Figure 1H). Although the freeze-and-thaw procedure can effectively remove microglial contamination, it is preferable to freeze the cultures before the rapid expansion of microglia to maintain higher astrocyte purity.

• 20.
  Remove the medium containing floating microglia and harvest primary astrocytes.

  • a.
    Wash the cells with pre-warmed HBSS three times and shake the flask to help remove microglia.
  • b.
    Treat adherent cells with 10 mL 1× Trypsin in HBSS for 15 min at 37°C.
  • c.
    Quench trypsin with 10 mL astrocyte culture medium.
  • d.
    Collect cells into a 50-mL tube.
  • e.
    Centrifuge at 200 × g for 5 min at room temperature (20°C −25°C).
  • f.
    Discard supernatant and resuspend the pellet in 10% DMSO in FBS (or designated storage medium).
• 21.
  Store primary astrocytes in freezing medium containing 10% DMSO in FBS.

  • a.
    Place the vials in a controlled-rate freezing container at −80°C for at least 24 h.
  • b.
    Transfer them to liquid nitrogen for long-term storage.

Note: In our experience, the primary astrocytes can store up to 3 months with a good survival rate (>80 %) after thaw from liquid nitrogen.

Note: For each flask (derived from the cortex of one mouse), prepare two vials for freezing, each containing approximately 2.5–3 × 10⁶ cells.

Astrocyte thawing and plating

Timing: 1 h

• 22.Coat 15 cm petri-dishes with 10 mL poly-L-lysine (0.1 mg/mL) for at least 1 hour and wash away with HBSS.
• 23.Add 10 mL of astrocyte culture medium to a 15-mL conical tube.
• 24.Load the 15 ml tube with 10 mL astrocyte medium.
• 25.
  Thaw frozen astrocytes rapidly in a 37°C water bath.

  • a.
    After thawing completely, slowly transfer the cell suspension into a 15 mL tube containing astrocyte culture medium.
  • b.
    Mix gently by inversion.

Note: Do not remove the freezing medium by centrifugation, as this can markedly reduce primary astrocyte viability.

• 26.Return the dishes/plates to a humidified incubator and incubate at 37°C with 5% CO₂.
• 27.After 24 h, replace the medium completely with fresh astrocyte culture medium. Maintain the culture by changing 50% of the medium twice per week.
• 28.
  On DIV21, primary astrocytes can be resuspended and plated for the design experiment.

  Note: When maintained under optimal conditions, the cells should reach ∼95% confluence and establish a consistent monolayer.

  • a.
    Wash the cells twice with 1× PBS.
  • b.
    Add 10 mL of 0.05% trypsin-EDTA in HBSS to the flask and incubate for 15 min at 37°C to detach the adherent cells.
  • c.
    Quench the trypsin by adding 10 mL of astrocyte culture medium.
  • d.
    Transfer the cell suspension to a 50-mL conical tube.
  • e.
    Centrifuge at 200 × g for 5 min at room temperature (20°C−25°C).
  • f.
    Discard the supernatant and resuspend the cell pellet in 1–2 mL of primary astrocyte culture medium.
  • g.
    Count the cells using Trypan Blue exclusion.
  • h.
    Adjust the cell concentration using astrocyte culture medium.
• 29.
  Plate the cells onto poly-L-lysine-coated culture vessels at the following densities:

  • a.
    1-2 × 10⁶ cells per 10-cm dish.
  • b.
    5 × 10⁵ cells per 6-cm dish.
• 30.Return the dishes/plates to a humidified incubator and incubate at 37°C with 5% CO₂.

Astrocyte activation with the cytokine mixture

Timing: 20 min, total 24 h

• 31.On DIV27, prepare the cytokine mixture and replace the existing medium with the cytokine-containing medium.

Note: Prepare the cytokine mixture fresh on the day of the experiment.

• 32.Wash the cells twice with pre-warmed HBSS.
• 33.Add the medium containing the cytokines mixture to the dishes or plates.
• 34.Return the dishes/plates to a humidified incubator and incubate at 37°C with 5% CO₂ for 24 h.

Protein precipitation and metabolite extraction by PCA

This section describes the workflow for harvesting primary astrocytes, extracting small-molecule metabolites using PCA, neutralizing the extract for UPLC-MS analysis, and recovering the protein pellet for total protein quantification. These steps generate metabolite samples suitable for UPLC-MS analysis and matching protein measurements for signal normalization.

Protein precipitation of primary astrocytes using 0.6N PCA

Timing: 2 h

• 35.Prepare 0.6 N PCA in a 2.3 mg/L Fenclonine (2.3 ppm Fenclonine) solution and keep it on ice until use.
• 36.
  Harvest primary astrocytes.

  • a.
    Carefully remove culture dishes containing primary astrocytes from the incubator.
  • b.
    Wash the cells twice with pre-warmed PBS.
  • c.
    Digest cells with 2 mL (6-cm dish) or 5 mL (10-cm dish) 0.05% trypsin-EDTA at 37°C for 10 min.
  • d.
    Quench the trypsin by adding an equal amount of astrocyte culture medium.
  • e.
    Collect the cell suspension, rinse the plate with colorless PBS, and combine the washes.
  • f.
    Centrifuge at 200 × g for 5 min at 4°C and carefully discard the supernatant.
  • g.
    Resuspend the pellet in 1 mL PBS and transfer to a 1.5-mL tube.
  • h.
    Centrifuge at 400 × g at 4°C and carefully discard the supernatant.

Repeat step 36-g and 36-h one more time.

Note: The suspension should be colorless as shown in Figure 2A to ensure that no culture medium remains.

Figure 2.

Critical extraction steps and targeted metabolite profiles in cytokine-activated primary astrocytes

(A) Image of the cell suspension prior to the addition of 0.6 N PCA.

(B) Image of the cell pellet after mixing with 0.6 N PCA, showing protein precipitation.

(C) Image of the protein pellet following centrifugation.

(D) Verifying pH value using pH test paper to confirm proper neutralization of PCA with K₂CO₃.

(E) Image showing CO₂ bubbling that occurs upon the addition of K₂CO₃ to the PCA extract.

(F) Image showing salt precipitation formed after the neutralization reaction.

(G) Imaging showing how polymers and salts affect the UPLC-MS signals. Left panel showing the ESI negative and right panel showing the ESI positive mode.

(H) Cytokine-induced changes in metabolite levels, including purinergic metabolites, glutamate-related intermediates, TCA cycle components, and glycolytic intermediates. Data are represented as mean ± SEM and analyzed using Student’s t test. ns, no significant; ∗, p < 0.05, ∗∗; p < 0.0005.

• 37.Remove any residual liquid completely.
• 38.Resuspend the pellet in 85 μL of pre-chilled 0.6 N PCA containing Fenclonine.

Note: Pipette thoroughly to ensure the pellet is fully resuspended. Protein precipitation should occur upon resuspension (Figure 2B).

• 39.Sonicate on ice for 5 min with alternating 30-second on and 30-second off intervals, ensuring the sample remains cooled throughout the process.
• 40.Centrifuge the samples at 11,000 × g for 30 min at 4 °C.
• 41.Carefully transfer 60 μL of the supernatant into a clean (non-sterilized) microcentrifuge tube and retain the pellet for subsequent protein analysis.

Note: Do not use sterilized micro centrifuge tubes, as autoclaving can release polymer contaminants that interfere with small-molecule detection in MS.

Note: Avoid aspirating any protein precipitate (Figure 2C), as this can adversely affect MS analysis and total protein content calculation.

• 42.Remove any remaining liquid from the pellet and proceed with protein pellet processing starting at step 54.

Neutralization of PCA extracts for UPLC-MS analysis

Timing: 3 h

• 43.
  Add 10 μL 1.8 M K₂CO₃ (0.6 N PCA: K₂CO₃, 6:1) quickly.

  Note: Rapid bubbling will occur immediately due to CO₂ release, and a white precipitate will form as salts crystallize during neutralization.

  Note: Before subjecting your sample to the procedure, we recommend to check the pH value using testing paper to ensure proper neutralization (Figure 2D).

  Note: Do not pipette after bubbling occurs to prevent the uneven mixing and avoid precipitated protein sticking to the pipette tips (Figure 2E).

  • a.
    Vortex briefly, spin down to collect the contents.
  • b.
    Open cap to release accumulated CO₂, and ensure that bubbling has completely stopped.
  • c.
    Vortex the samples again to mix thoroughly.
  • d.
    Place the tubes on ice for 10 min.
  • e.
    Open the cap to allow complete venting of any remaining CO₂.
• 44.Centrifuge at 11,000 × g for 30 min at 4°C.
• 45.Transfer 60 μL of the clear supernatant to a new non-sterile tube.

Note: Do not disturb or transfer the salt pellet (Figure 2F), as carryover of the precipitation may interfere with MS signal during analysis.

• 46.Remove the solvents using a SpeedVac vacuum concentrator prior to LC-TOF analysis.

Note: Drying time will vary depending on the sample number and volume. Complete evaporation typically requires 1–2 h.

• 47.After the samples dry completely, add 100 μL of ACN:MeOH:ddH₂O (2:2:1, v/v/v) to each tube for reconstitution.
• 48.Vortex each sample for ∼10 s to initiate mixing.
• 49.Place the tubes in an ice-bath sonication device and sonicate for 5 min continuously to ensure complete reconstitution.
• 50.Centrifuge the samples at 16,110 × g for 10 min at 4°C.
• 51.Transfer the clear supernatant into LC-MS glass vials with insert tubes and caps, and keep it at 4°C.
• 52.Prepare a QC sample by mixing equal aliquots (5–10 uL) from each individual sample into a single pooled tube.
• 53.Before analyzing the experimental samples, inject the QC sample six consecutive times to stabilize the LC-MS system and ensure retention-time consistency.

Note: This step can help to minimize peak shift and improves data reproducibility.

Resolubilizing protein pellets for protein quantification

Timing: 3 h

• 54.Add 500 μL of ice-cold 100% acetone containing 20 mM DTT to the pellet. Mix by vortexing.
• 55.Incubate the samples at −20°C for at least 1 hour or overnight.
• 56.Wash the pellet by vortexing three times for 10 s each.
• 57.Centrifuge at 15,000 × g for 15 min at 4°C. Discard the supernatant.
• 58.
  Add 500 μL ice-cold 80% acetone to wash the protein pellet.

  • a.
    Vortex for 30 s.
  • b.
    Centrifuge at 15,000 × g for 2 min and discard the supernatant.
• 59.Repeat step 58 twice more (for a total of three acetone wash cycles).

Note: The pellets can easily shift or slide when discarding the acetone, so handle the tube carefully to avoid losing material.

• 60.Air-dry the pellet for 5 min in a chemical hood.
• 61.Dissolve the pellet in 50–100 μL ice-cold 2-D sample buffer.
• 62.
  Freeze-thaw cycle.

  • a.
    Place the sample at −20°C and incubate for 1 hour to ensure complete freezing.
  • b.
    Transfer the sample to 4°C and allow it to thaw with intermittent shaking for 15 min.
• 63.Sonicate the sample for six cycles of 10 s each.

Note: Keep the samples on ice during sonication and avoid generating foam.

• 64.Centrifuge the sample at 6,000 × g for 10 min.
• 65.Carefully collect the supernatant without disturbing any residual material at the bottom of the tube.

Note: After complete solubilization, the sample should appear clear with no visible pellet remaining.

• 66.Quantify the protein concentration using the Bio-Rad Bradford assay kit and calculate the total protein content.

Note: For our experimental conditions, each 6-cm dish of primary astrocytes typically yields approximately 70–120 μg of total protein.

Data analysis

Quantify targeted metabolites from UPLC-MS data

Timing: 1 h

This section describes how to process UPLC-MS output, extract metabolite peak areas, and normalize metabolite signals to the internal standard and total protein amount. These steps provide relative metabolite levels for downstream comparison and statistical analysis.

• 67.After completing UPLC-MS analysis, extract ion chromatograms corresponding to each targeted metabolite from the raw data using the instrument software (e.g., Agilent Profinder B.10.00), and integrate the peak areas.
• 68.
  Determine relative quantification by dividing the peak area of each metabolite by that of the internal standard (Fenclonine) multiplied by the total protein amount of each sample.

  • a.
    Retrieve the total protein amount (μg) measured from the corresponding protein pellet of each sample (from step 66).
  • b.
    Normalize metabolite signals by dividing the integrated peak area (AUC) of each metabolite by both the total protein amount (μg) and the peak area of the internal standard, Fenclonine.
• 69.Calculate the Normalized Metabolite Signal (representing the final relative metabolite level used for statistical analysis) using the following equation:

$$\text{Normalized}\text{Metabolite}\text{Signal}=\frac{\text{Integrated}\text{peak}\text{area}\left(AUG\right)\text{metabolite}}{\text{Integrated}\text{peak}\text{area}\left(AUG\right)\text{Fenclonine}\times \text{Total}\text{protein}\text{amount}\left(\mu G\right)}$$

Expected outcomes

Upon successful completion of this protocol, a highly enriched population of primary astrocytes should be obtained, as illustrated in Figures 1H and 1I. Astrocyte purity can be verified by immunostaining for GFAP, a canonical astrocytic marker (Figure 1I). In addition, quantitative PCR (qPCR) analysis of astrocyte-enriched genes (e.g., Glul, Slc1a3, and Aqp4), along with minimal expression of microglial (Aif1), neuronal (Tubb3), oligodendrocyte (Mbp) and NG2 cell (Cspg) markers, can be used to further confirm culture purity (Figure 1J). Successful induction of astrocyte reactivity following cytokine treatment should be evident by significant upregulation of reactive astrocyte–associated genes as measured by qPCR (Figure 1K).

For metabolite extraction and mass spectrometry analysis, successful sample preparation should yield clean and stable electrospray ionization (ESI) signals (Figure 2G, top). In contrast, polymer or salt contamination can suppress metabolite ion signals and can be recognized by changes in the total ion chromatogram (TIC). Samples containing such contaminants (Figure 2G, middle and bottom) exhibit altered TIC profiles compared with uncontaminated samples (Figure 2G, top). A robust and consistent Fenclonine internal standard signal indicates efficient and uniform metabolite extraction across samples. Following cytokine stimulation, alterations are expected in purinergic metabolites and glutamate cycling pathways, whereas moderate changes are typically observed in TCA cycle and glycolytic intermediates (Figure 2H).

Limitations

A key limitation of this protocol is that it does not provide guidance on UPLC-MS instrument setup, tuning, or calibration. These procedures are highly instrument-dependent and subject to variation across different platforms, column chemistries, and ion source configurations. As a result, users must optimize these parameters independently in accordance with their specific instrumentation, facility standards, and manufacturer recommendations.

This protocol describes the quantification of targeted metabolites, with a focus on adenosine-related metabolites, glycolytic intermediates, and selected TCA cycle substrates. Although other metabolites can be measured using the procedures outlined here, not all metabolites are optimally detected under the current extraction and instrument settings. As demonstrated in Table 1, the same metabolites can show different signal intensities when extracted with 0.6 N PCA versus ACN/MeOH -based methods, highlighting method-dependent variation in recovery and detection. We therefore recommend performing preliminary optimization to determine the most suitable extraction and detection conditions for the specific metabolites of interest before processing experimental samples.

Table 1.

Comparison of signal intensities between PCA and ACN/MeOH extraction methods

Compound name	Formula	Mass	Cas ID	PCA extraction	ACN/MeOH extraction
Fenclonine	C9 H10 Cl N O2	199.038	14173-39-8	39992	17754
Itaconic acid	C5 H6 O4	130.026	97-65-4	N.D	4267
alpha-KG	C5 H6 O5	146.021	328-50-7	3799	26789
Inosine	C10 H12 N4 O5	268.080	58-63-9	4142	16916
ATP	C10 H16 N5 O13 P3	506.995	56-65-5	149355	339574
Adenine	C5 H5 N5	135.055	73-24-5	7697823	366604
Adenosine	C10 H13 N5 O4	267.097	58-61-7	176322	45343

The TCA cycle is a highly dynamic metabolic pathway, and although astrocytes are often considered more glycolytic than neurons, TCA cycle intermediates can still be challenging to detect reliably. In our hands, several TCA substrates—including 2-HG, citrate, succinate, and malate—can be consistently detected using this protocol. However, other intermediates are not detected as robustly under the current extraction conditions and instrument configuration. If comprehensive TCA cycle profiling is a major focus of your study, we recommend incorporating an additional derivatization step using O-benzylhydroxylamine (O-BHA) and considering alternative chromatographic separation (e.g., an ACQUITY UPLC BEH C18 column) to improve detection sensitivity and metabolite coverage.¹³

Although this protocol demonstrates that precipitated proteins can be resolubilized for total protein quantification, their use for downstream expression analyses is limited. In our experience, several astrocytic proteins exhibit altered banding patterns and apparent expression levels after undergoing the precipitation–resolubilization procedure (Figure 3A). These changes likely result from protein denaturation or partial loss during the extraction and resolubilization steps. Therefore, we do not recommend using resolubilized protein samples to assess differential protein expression by western blotting or similar assays. Users should carefully evaluate potential alterations in protein integrity before applying this method to expression-based analyses.

Figure 3.

Western blot comparison of protein expression from precipitation–resolubilized samples versus RIPA-extracted protein lysates

(A) Western blot analysis of mitochondrial proteins and astrocyte-enriched proteins comparing precipitation–resolubilized samples with RIPA-extracted protein lysates.

In this protocol, Fenclonine is used as the internal standard because it is non-endogenous to mammalian cells, cost-effective, commercially accessible, and exhibits stable retention under both amide and C18 chromatographic conditions.^14,15,16 While stable isotope–labeled metabolites can provide improved quantitative accuracy, their higher cost and limited availability may restrict routine use in some laboratories. Therefore, this protocol prioritizes practicality and broad applicability for cell biology laboratories. Researchers requiring absolute quantification or enhanced analytical precision may consider incorporating stable isotope–labeled standards, such as ¹³C-labeled AMP or ATP,¹⁷ as appropriate.

Troubleshooting

Problem 1

Primary astrocytes display low viability (<50% at 24 h post-thaw) and poor proliferation following thawing (step 27).

Potential solution

In our experience, primary astrocytes show markedly reduced post-thaw viability after extended storage, even when preserved in liquid nitrogen. To improve recovery, use frozen stocks within 3 months, ensure proper cryopreservation with 10% DMSO and controlled-rate freezing, avoid repeated freeze–thaw cycles, and rapidly thaw the vial in a pre-warmed water bath to promote maximal post-thaw survival and proliferative capacity. When transferring thawed cells into culture medium, do so slowly to minimize osmotic shock. Do not vortex or pipette the thawed cells, as mechanical stress can cause significant damage. Instead, gently invert the tube several times to mix. Avoid storing cells directly in culture medium, as this greatly reduces viability. We have not evaluated commercial cryopreservation solutions. However, some products report improved post-thaw survival compared with standard 10% DMSO.

Problem 2

Low metabolite signal (step 67).

Potential solution

Confirm that all residual PBS is completely removed before adding PCA (step 37), as leftover salts can suppress ionization and substantially affect MS signal. After PCA neutralization with K₂CO₃, do not disturb or transfer the salt pellet (step 45), as carryover of the precipitate can interfere with MS detection. Ensure that you are not using consumables sterilized by high-pressure autoclaving (e.g., pipette tips, microcentrifuge tubes), and avoid using autoclaved ddH₂O, as these can introduce small contaminating compounds that interfere with UPLC-MS detection. Additionally, some metabolites may not be optimally extracted or detected under the conditions described in this protocol. We recommend performing preliminary optimization—such as testing ACN/MeOH/H₂O extraction or alternative derivatization procedures—particularly if you are using a different mass spectrometry platform or source configuration.

Problem 3

Uneven Fenclonine signal across samples within the same batch (step 68).

Potential solution

During the neutralization step, add K₂CO₃ quickly to ensure complete reaction with the PCA extract (step 43). Avoid pipetting once foaming and salt precipitation occur, as further agitation at this stage can lead to incomplete neutralization and variability in internal standard recovery. Allow CO₂ to vent completely by opening the tube lids for more than 5 s (step 43b and 43e) to ensure full gas release and consistent handling across all samples.

Resource availability

Lead contact

Further information and requests for resources should be directed to and will be fulfilled by the lead contact, Yijuang Chern (bmychern@ibms.sinica.edu.tw).

Technical contact

For questions regarding experimental procedures, protocol implementation, or technical details, please contact the technical contact, Ya-Gin Chang (yaginchang@ibms.sinica.edu.tw).

Materials availability

This study did not generate new unique reagents.

Data and code availability

• •No new reagent was generated in this study.
• •No new code was generated in this study.
• •Any additional information required to reanalyze the data reported here is also available from the lead contact.

Acknowledgments

This study was supported by the National Science and Technology Council of Taiwan (MOST 110-2321-B-001-011, MOST 111-2321-B-001-010, NSTC 112-2321-B-001-008, and NSTC 113-2321-B-001-011). We thank Teng-Yang Hsu for assistance with collecting photographs documenting the procedures for P1 mouse collection, dissection, and tissue homogenization.

Author contributions

Y.-G.C. developed and optimized the protocol, performed the experiments, and wrote the manuscript. G.-M.L. and C.-Y.L. contributed to protocol development and optimization and provided technical support. Y.C. supervised the project and critically revised the manuscript.

Declaration of interests

The authors declare no competing interests.

Declaration of generative AI and AI-assisted technologies in the writing process

During the preparation of this manuscript, the authors used ChatGPT to improve language and readability. After using this tool, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication.