Microscopy-based protocol for the quantification of cells viability for temperature-sensitive S. pombe

Summary

Conventional colony-forming unit assay to measure cell viability is laborious and results in large experimental variability, which prohibits accurate quantification of microbial viability. Here, we present a microscopy-based protocol for the quantification of cells viability for temperature-sensitive S. pombe. We describe steps for growing and treating yeast cells and visualization of individual cells viability based on Phloxine B staining. We then detail procedures for data processing using Nikon NIS Elements Advanced Research (AR) software.

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

Graphical abstract

Highlights

• •Instructions for reviving cryopreserved yeast stock onto the desired growth medium
• •Steps and conditions for growing temperature-sensitive yeast cells
• •Steps for visualizing individual cells viability based on Phloxine B staining

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

Conventional colony-forming unit assay to measure cell viability is laborious and results in large experimental variability, which prohibits accurate quantification of microbial viability. Here, we present a microscopy-based protocol for the quantification of cells viability for temperature-sensitive S. pombe. We describe steps for growing and treating yeast cells and visualization of individual cells viability based on Phloxine B staining. We then detail procedures for data processing using Nikon NIS Elements AR software.

Before you begin

Essential genes are indispensable for cell survival, hence, to study their loss of function effect on cell, conditional temperature-dependent loss of function mutation is usually incorporated to these genes. The proteins encoded by these mutated genes will lose their function upon temperature shift. The protocol described herein can be applied to compare the viability of these mutants at both permissive and restrictive temperatures.

Here, we use the temperature sensitive (ts) mutant of cnp1⁺ gene that expressed the fission yeast Schizosaccharomyces pombe CENP-A (SpCENP-A)—the centromere-specific histone H3 variant—to demonstrate the steps involved in this protocol. The mutant (named cnp1-1) contains a missense L87Q (number 87^th leucine is mutated to glutamine) mutation incorporated to express the mutant SpCENP-A^cnp1-1 protein. The SpCENP-A^cnp1-1 delocalizes from centromere when subject to high temperature (33°C–36°C).² Centromeric delocalization of the mutant SpCENP-A^cnp1-1 will subsequently lead to chromosome segregation defect and cell death at such non-permissive temperature. We recently reported that knocking out of the set2⁺ gene that encodes histone H3 lysine 36 (H3K36) methyltransferase (Δset2) to suppress the ts growth defect of cnp1-1 mutant using spotting assay.¹ We subsequently demonstrated and quantified the viability difference of cnp1-1 and Δset2cnp1-1 strains using current protocol.

The advantage of this protocol over the colony-forming unit (CFU) assay is a higher accuracy in quantification as individual cells are accountable under microscopy observation for viability determination, as the dead cells are clearly stained. In contrast, CFU assay is a multistep process, involving determining cell concentration using hemacytometer or cell counter, dilution and plating of fixed number of cells onto agar plate. Any error that occurs in one of the aforementioned steps will magnify the difference between the strains tested. Hence, the resolution to accurately differentiate strain viability is low, particularly from those with minor differences.

Before starting, the growth rate of the strain in the respective growth medium and incubation temperature should be preliminarily identified. The protocol below can be modified to quantify viability of yeast cells undergoing other treatments, such as addition of DNA damaging agents, inhibitors and to compare effects of mutations or gene expression on cells of various genetic backgrounds. Factors such as duration of treatment, concentration or intensity of agent used, temperature of incubation and the starting cells concentration should be identified with a pilot test using positive and negative control strains. This is because each agent has their own optimal conditions to achieve maximum effectiveness.

Preparation of growing media (agar plate)

Timing: 3 days

To revive frozen cell stock from freezer, prepare Yeast Extract Adenine (YEA) agar plate. YEA is a rich medium suitable for the growth of a wide spectrum of S. pombe cells. The media used will be dependent on the need of each experiment. YES (compositions) rich media can also be used in the case of S. pombe.

• 1.In a 1 L bottle, prepare YEA according to the recipe below.
• 2.After adjusting to pH 5.5 using 2 N HCl, add 20 g/L Bacto agar.

Note: pH 5.5 is essential for the optimal growth of S. pombe.

• 3.Add in magnetic stir bar and autoclave for 121°C 20 min.
• 4.Cool to around 50°C while stirring on the magnetic stirrer or until the bottle can be held by hand.
• 5.Pour the liquid onto sterile petri dish until the solution covered the bottom of the dish (∼20 mL) and leave to set.

Note: Ensure the place where pouring agar solution is properly clean with 70% ethanol.

• 6.Once the color of the plate turns from amber to light brown, invert the plate to prevent condensation on the agar surface.
• 7.Leave at room temperature (23°C–26°C) for at least 3 days to dry before use.

Revive and grow frozen yeast stock

Timing: 10 min

• 8.Use a sterile pipette tip, scrape off a layer of frozen cultured previously stored (resuspended in 20% glycerol) in the −80°C freezer.

Note: The tube storing the frozen culture should not be left at room temperature for long period of time to avoid thawing.

• 9.Place the culture onto a YEA agar plate, leave to dry.
• 10.Once dry, incubate the plate in inverted position at 26°C incubator for 3 days.

Note: Ensure the incubation temperature is optimal for the strain.

• 11.The cells will grow and form a white patch, indicating they are ready to be used.

Preparation of growing liquid media

Timing: 3–4 h

• 12.In a 1 L bottle, prepare YEA according to the recipe below.
• 13.Adjusting to pH 5.5 using 2N HCl.

Note: pH 5.5 is essential for the optimal growth of S. pombe.

• 14.Autoclave for 121°C 20 min.

Note: Alternatively, the liquid media can be filter sterilized using a 0.45 μm filter unit.

• 15.Cool to room temperature (23°C–26°C) before use.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Experimental models: Organisms/strains
Wild-type h+leu1-32	Lim et al.1	SPKK317
Δset2 (h+set2::kanMX6 leu1-32)	Lim et al.1	SPKK316
cnp1-1 (h+cnp1::ura4 cnp1-1[lys1+] ura4::D18 leu1-32)	Chen et al.2	Cnp1-1
Δset2cnp1-1 (h-set2::kanMX6 cnp1::ura4 cnp1-1[lys1+] ura4::D18 leu1-32)	Lim et al.1	SPKK354
Chemicals, peptides, and recombinant proteins
Phloxine B	Nacalai Tesque Inc	CAS: 18472-87-2
Sodium chloride	Sigma-Aldrich	CAS: 7647-14-5
Potassium chloride	Sigma-Aldrich	CAS: 7447-40-7
Sodium phosphate dibasic	Sigma-Aldrich	CAS: 7558-79-4
Potassium phosphate monobasic	Sigma-Aldrich	CAS: 7778-77-0
Hydrochloric acid 37%	Sigma-Aldrich	CAS: 7647-01-0
Adenine	Sigma-Aldrich	CAS: 73-24-5
D-(+)-Glucose	Sigma-Aldrich	CAS: 50-99-7
Bacto yeast extract	BD Biosciences	Cat#: 212750
Bacto agar	BD Biosciences	Cat#: 214010
Absolute ethanol	Fisher Chemical	CAS: 64-17-5
Immersion oil for microscopy	Nikon	Cat#: MXA20235
Software and algorithms
Nikon NIS Elements AR (v5.02)	Nikon	https://www.microscope.healthcare.nikon.com/en_AOM/products/software/nis-elements/nis-elements-advanced-research
Microsoft Office 365–Excel	Microsoft	https://www.microsoft.com/en-us/microsoft-365/excel
Other
P20 PIPETMAN	Gilson	Cat#: F123600
P200 PIPETMAN	Gilson	Cat#: F123601
P1000 PIPETMAN	Gilson	Cat#: F123602
Innova 44R refrigerated incubator shaker (22°C/33°C)	Eppendorf	Cat#: M1282-0006
26°C incubator	Eyela	Cat#: LTI-700
Ultra-low temperature freezer (−80°C)	PHCbi	Cat#: MDF-DU702VHL
Sorvall Legend micro 21R refrigerated centrifuge (4°C)	Thermo Fisher Scientific	Cat#: 75002447
Ultrospec spectrophotometer	Biochrom	Cat#: 80-2116-30
Autoclave	ALP	Cat#: CL-40M
Magnetic stir bar (40 mm)	VWR	Cat#: VWRI442-4527
Magnetic stirrer (Thermo Cimarec+ stirring hotplates)	Thermo Fisher Scientific	Cat#: SP88857107
1 L bottle	Schott DURAN	Cat#: SCOT218015455
Eclipse Ti-E inverted microscope	Nikon	https://www.microscope.healthcare.nikon.com/en_AOM/products/inverted-microscopes/eclipse-ti-series
100x oil immersion objective lens	Nikon	Model: CFI PLAN APO VC 100XH 1.4 NA
ET-mCherry filter	Chroma	Cat#: 490008
ORCA-Flash2.8 digital camera (for microscope)	Hamamatsu	Cat#: C11440-10C
Pipette tips (200 μL)	Greiner	Cat#: 775350
Pipette tips (1,000 μL)	Greiner	Cat#: 777350
Microcentrifuge tube (1.5 mL)	Eppendorf	Cat#: 0030120086
Centrifuge tube (50 mL)	Greiner	Cat#: T2318
Microscope slide	Continental Lab Products	Cat#: 7105
Microscope cover glasses (22 × 22 mm)	Paul Marienfeld GmbH & Co	Cat#: 0101050
Petri dish (90 × 15 mm)	Biomedia	Cat#: 990000PQ
Kimwipes	Kimtech Science	Cat#: 34155
0.45 μm filter unit	Corning	Cat#: 431206
0.22 μm syringe filter	Sartorius	Cat#: 16532-k

Note: Strains stated in this table can be substituted with strains user intend to study on.

Note: All chemicals and reagents can be substituted using similar products of other brands.

Materials and equipment

Phosphate-Buffered Saline (PBS) 1x (1 L)

Reagent	Final concentration	Amount
Sodium chloride	137 mM	8 g
Potassium chloride	2.7 mM	0.2 g
Sodium phosphate dibasic	10 mM	1.42 g
Potassium phosphate monobasic	1.8 mM	245 mg
MiliQ water	N/A	To make 1 L
Total	N/A	1 L

Storage: Long-term at 4°C.

Note: Adjust to pH 7.4 using 2N HCl and autoclave.

70% Ethanol (100 mL)

Reagent	Final concentration	Amount
Absolute ethanol	70%	70 mL
ddH2O	N/A	30 mL
Total	N/A	100 mL

Storage: Long-term at room temperature (23°C–26°C).

CRITICAL: Keep the ethanol stock away from potential fire source.

2N Hydrochloric acid (HCl) (50 mL)

Reagent	Final concentration	Amount
37% Hydrochloric acid	2 N	8.212 mL
MiliQ water	N/A	41.788 mL
Total	N/A	50 mL

Storage: Long-term at room temperature (23°C–26°C).

Note: In the fume hood, add HCl to the water slowly (caution: not vice versa).

CRITICAL: The stock HCl is a strong acid with highly corrosive even in vapor form, hence only open and dispense the stock in fume cabinet. Wear double gloves and the required PPE.

Yeast extract adenine (YEA) media (1 L)

Reagent	Final concentration	Amount
Yeast extract	N/A	5 g
Adenine	0.56 mM	75 mg
Glucose	166.5 mM	30 g
MiliQ water	N/A	To make 1 L
Total	N/A	1 L

Storage: Use fresh or short-term storage (1 month) at room temperature (23°C–26°C).

Note: Adjust to pH 5.5 using 2N HCl. Autoclave or filter sterilize (using 0.45 μm filter unit).

Yeast extract adenine (YEA) agar plate (1 L)

Reagent	Final concentration	Amount
YEA media (before autoclave)	N/A	1 L
Bacto Agar	N/A	20 g
Total	N/A	1 L

Storage: Use fresh or short-term storage (1 month) at 4°C.

Note: Autoclave, cool down and pour into petri dishes. Invert the petri dishes when agar solidified.

Phloxine B stock (1 mL)

Reagent	Final concentration	Amount
Phloxine B	N/A	1 mg
ddH2O	N/A	1 mL
Total	N/A	1 mL

Storage: Use fresh or short-term storage (1 month) at 4°C.

Note: The stock solution should be filter-sterilized using 0.22 μm syringe filter and store in 4°C before usage.

Step-by-step method details

Growing of yeast cells

Timing: 3 days

This section describes the reviving of cryopreserved yeast stock onto the desired growth medium, allowing them to recover, followed by inoculation into a larger culture.

• 1.
  Pre-inoculation of fission yeast cells.

  • a.
    Pick a loopful of cells using sterile pipette tips from the agar plate into 5 mL of YEA media placed in a 50 mL centrifuge tube.

    Note: Alternatively, capped tubes and Erlenmeyer flask can be used. 50 mL tubes are used to culture 5 mL of cells to provide the space for sufficient mixing and aeration and to minimize the sedimentation of the yeast cells to the bottom of the tubes during overnight culturing. The 50 mL tubes should be placed at an angle to facilitate efficient mixing of the liquid content without spillage. Similar consideration of proper mixing to aerate the cultures should also be considered using capped tubes and Erlenmeyer flasks. If the culture volume is smaller or larger, size of the tubes or flasks can be reduced and increased accordingly.

  • b.
    Shake the cells overnight at 22°C in a shaking incubator at 110 rpm.
• 2.
  Dilution of cells.

  • a.
    Next day (from step 1b), measure the optical density at 600 nm (OD) using spectrophotometer.
  • b.
    Dilute the cells to OD 0.125 in 20 mL of YEA placed in a 50 mL centrifuge tube.

    Note: Alternatively, capped tubes or Erlenmeyer flask can be used.

  • c.
    Shake the cells overnight at 22°C in a shaking incubator at 110 rpm.

    Note: Assuming cells doubling time at 22°C is 6 h. 12 h later (overnight) will be around log phase of cells (∼OD 0.5). The duration of incubation depends on the intention start time the next day. Different strains might have different doubling time and optimum incubation temperature.

Treatment of cells

Timing: 48 h

This section describes the growth conditions, volume and number of cells needed for each treatment.

• 3.
  Preparation of non-treated cells.

  • a.
    Measure the OD of cells.

    Note: Desired OD is 0.5, but OD < 1.0 is in acceptable range.

  • b.
    Aliquot 10 mL of cells (OD ∼0.5) from previous day and proceed to Step 5. This sample will be labeled “22°C” (Non-treated).

    Note: If OD exceeded 0.5, the culture can be diluted to OD 0.5 in 10 mL.

• 4.
  Preparation of cells with treatment (See troubleshooting 1).

  • a.
    Diluted the remaining cells to OD 3 × 10⁻⁸ (by calculation) in 20 mL of fresh YEA media placed in a 50 mL centrifuge tube.

    Note: Alternatively, capped tubes or Erlenmeyer flask can be used.

  • b.
    Shake the cells for 48 h at 33°C in a shaking incubator at 110 rpm.

    Note: Assuming cells’ doubling time at 33°C is 2 h. 48 h later will be around log phase of cells (∼OD 0.5).

  • c.
    Samples will then proceed to Step 6 and labeled as “33°C” (treated).

Staining of cells

Timing: 1.5 h

This section describes the procedure of cell staining using Phloxine B, followed by a washing procedure to remove excess stain.

• 5.
  Staining of non-treated cells (See troubleshooting 2).

  • a.
    For cells from Step 3b, add 5 μL of Phloxine B stock into 10 mL of cell culture (final concentration of 5 μg/mL) and continue shaking in 22°C incubator for 1 h.
  • b.
    Proceed to Step 7 thereafter.
• 6.
  Staining of treated cells (See troubleshooting 2).

  • a.
    For cells from Step 4c, at the 47^th h of 33°C incubation, add 5 μL of Phloxine B per 10 mL cell culture and continue shaking in 33°C for 1 h.
  • b.
    Proceed to Step 7 thereafter.
• 7.
  Washing of stained cells.

  • a.
    Centrifuge 1 – 2 mL (OD 0.5) of cell culture inside a 1.5 mL microcentrifuge tube at 2400 × g for 1 min.
  • b.
    Pour away the supernatant, quick spin using centrifuge and remove residual supernatant using pipette.

    Note: Alternatively, an aspirator can be used. Ensure aspiration of supernatant is done immediately after centrifugation to avoid loosening of the cell pellet.

  • c.
    Add 1 mL cold 1x PBS and resuspend to wash the cell pellet.
  • d.
    Centrifuge for 2400 × g for 1 min at 4°C and remove all supernatant.
  • e.
    Repeat Steps 7c-d twice.
  • f.
    Resuspend the cell pellet in 20 μL of 1x cold PBS and the cells are ready to be viewed under microscope.

    Note: Proceed to image acquisition step immediately once cells are ready.

Image acquisition

Timing: variable (depends on number of samples)

This section describes the procedure to prepare cells for microscopy imaging and steps needed for automated image acquisition.

• 8.
  Mounting samples onto the microscope slide (See troubleshooting 3).

  • a.
    Wipe microscope slide and cover glasses with 70% ethanol and Kimwipes to remove any debris.
  • b.
    Place 2 μL of cell mixture from Step 7f to the center of microscope slide and place a cover glass on it.

    Note: No sealing of the slides is required as the sample is visualized immediately after mounting.

  • c.
    Place a drop of immersion oil at the center of cover glass and mount the slide on the microscope slide holder.
  • d.
    Observe the S. pombe cells with a 100x magnification objective lens using a fluorescence microscope.

    Note: We use the Nikon Eclipse Ti-E microscope system with NIS-Elements Advance Research software (v5.02) as an example.

• 9.
  Automatic acquisition of images (See troubleshooting 4).

  • a.
    Set up an automated image acquisition parameter (ND Acquisition) as shown in Figure 1.

    Note: it will first capture a still image of DIC then automatically switch to mCherry channel to capture Phloxine B signal of the same field of view and plane.

  • b.
    Select the destination where the file will be saved to.
  • c.
    Focus the cells by viewing the DIC image.
  • d.
    Then click the “Run now” button for automated image acquisition.
  • e.
    The captured images will be saved to a pre-destined folder automatically.
• 10.Capture number of images that will give a total number of cells > 200 per sample. (See troubleshooting 5).

Note: All Phloxine B-stained strains in the same experiment need to have images taken immediately after staining to ensure consistency.

• 11.Images of DIC and mCherry will be saved as a single .nd2 file format.

Note:Figure 2A showing an example capture of S. pombe cells under DIC and mCherry channel respectively and a combined image of both channels.

Figure 1.

Automated ND acquisition of multiple channels

First, to set the path where the file will be saved in. Then, under “Lambda” tab, tick the channel needed. Finally, once the image is in focus, press “Run now” to capture.

Figure 2.

Example of an .nd2 file saved

(A) The file contains images from DIC and mCherry channel, (B) The tab below labeled “All” merge both channels together.

Image processing

Timing: 1 h

This section describes how to combine microscope images of different channels into a single image.

• 12.To combine the images from both DIC and mCherry channel into a single image, open a .nd2 image using Nikon NIS Elements AR (v5.02), click on the “All” tab (Figure 2B).
• 13.To extract image of individual channel, navigate to the different tab below the image (All, DIC and mCherry). Click “File> Save as” to save the selected channel in the file type needed.

Data analysis

Timing: 1 h

This section describes quantification of cells viability using images from the “image processing” step.

• 14.In Nikon NIS Elements AR (v5.02) software, open the image with DIC and mCherry channel combined, count cells and record numbers of cells with and without red stain (example images are shown in Figure 3).
• 15.To determine the survival of cells under different temperature/treatment, use the following equation:

Figure 3.

Merged microscopic images between DIC and mCherry channel

Cells of different genetic background were treated at 22°C and 33°C, and stained with Phloxine B. Visually, large number of cnp1-1 cells were stained red by Phloxine B but obviously decrease in number of red-stained cells when set2 is concurrently deleted in cnp1-1. Bar, 20 μm.

Non-red stained cells / Total number of cells ∗ 100% = viability in percentage (%).

• 16.Repeat the experiment twice to obtain standard deviation.
• 17.Plot graph of viability (%) over strains (for example Figure 4).

Figure 4.

Quantification of cells viability of strains treated at 22°C and 33°C

Results reflected the spot assay shown in Figure 5, in which cnp1-1 inability to survive healthily at 33°C but can be partially rescued when set2 is deleted. N = 200.

Expected outcomes

Spotting assay is usually applied to test the growth difference between mutant strains in response to different treatments, but the difference is usually in a non-quantitative manner. Here, using four S. pombe strains as example, we show that the proposed protocol is able to give similar results that matched the spotting assay but in a quantitative manner. cnp1-1 is the mutant of centromere protein CENP-A, with a L87Q point mutation that confers delocalization of the mutant CENP-A protein from centromere at non-permissive temperature, which in turn leads to cell death associated with missegregation of chromosomes. Deletion of set2 in cnp1-1 strain background partially suppresses the temperature sensitive defects of cnp1-1.¹

In spotting assay, cnp1-1 strain shows sensitivity towards restrictive temperature 33°C (shown by 3 clear spots growing up at 33°C compared to WT with 7 spots) while deletion of set2 in cnp1-1 suppresses this growth retardation at 33°C (Figure 5). Hence using our method here, we expect to see the viability for cnp1-1 to be the lowest among these strains at 33°C followed by Δset2cnp1-1 and both WT and Δset2. As expected, using our Phloxine B staining method, we obtained results that reflect the spotting assay. Only about 49.5% of cnp1-1 cells survived at 33°C while the number increased when set2 was deleted in cnp1-1 to 74% (Figures 3 and 4).

Figure 5.

Knockout of set2 (Δset2) suppressed temperature sensitivity of cnp1-1 mutant

five-fold serially diluted samples were spotted on YEA agar plate and incubated at 26°C and 33°C for 3 days.

Limitations

Several issues or potential limitations should be considered before the start of experiment. First, this assay is effective in determining cells that lost viability but not those with non-lethal defects. Second, this protocol still involves manual counting of cells stained/unstained. Third, the conditions in this protocol (incubation temperature, duration, and staining time) are optimized to test on temperature-sensitive strain. Hence the changes in condition, for example, higher temperature use, the incubation duration might change accordingly. Fourth, this assay is suitable to test on small number of strains rather than big scale because of the involvement of multiple steps such as staining, washing and microscopic observation. Longer period spent on each step may affect the accuracy of the assay. Lastly, compared to CFU assay, this assay although more accurate (down to single cell), requires a fluorescence microscope with suitable light filters to view Phloxine B-stained cells.

This protocol can be adapted to be used for treatments other than temperature stress. Treatments such as with DNA damaging agent, UV radiation, nutrient deprivation, and osmotic pressure etc, can also be applied to cells. However, for different treatments, the duration of cell incubation or treatment, the temperature, and the starting concentration of cells must be optimized first. A spotting assay should be carried out beforehand with proper positive and negative controls to independently verify the result obtained by Phloxine B staining.

Troubleshooting

Problem 1

The result does not match the spot assay. (Step 4).

Potential solution

This protocol is optimized to be used on temperature sensitive strain that shows lost in viability when grown in restrictive temperature. Several factors must be considered before start if other treatment or non-temperature dependent strain is to be used.

• •Types of treatment: the effect of treatment varies greatly. DNA damaging drug may kill cells easily compared to temperature sensitive cells growing at restrictive temperature. Hence, might not need long time to cause cell death.
• •Concentration or intensity of treatment: Higher drug concentration or higher temperature will also be more effective in killing cells.
• •Duration of incubation: Depends on type of treatment, increase the intensity of treatment will result in faster killing, hence the incubation time should be shortened.
• •Starting OD: The initial OD should be calculated so that the cells would not be saturated at the end of the treatment (before staining). This is to avoid the survived/resistant cells to overgrow the death cell, masking the real viability count.
• •Temperature of incubation: Temperature used should be optimal for the cell or DNA damaging agent. Some chemicals are more effective at higher temperatures while some will lose their effectiveness. Always read the chemical specification before the start of experiments.

Problem 2

Most of the cell-stained red, includes WT cells. (Steps 5 and 6).

Potential solution

This might indicate the problem of overstaining. If different treatment (other than temperature) was used, do a preliminary study on the staining duration to optimize the staining efficiency with proper control in place.

Problem 3

Cells overlapping with each other or floating under microscope. (Step 8).

Potential solution

This may be due to the improper mounting of cells on microscope slide, or the resuspended cells are too concentrated. To eliminate the potential problem.

• •Avoid adding more than 2 μL of cell culture per slide.
• •Dilute the cells with 1x PBS if cell culture appears to be concentrated (hard to pipette).
• •Clean both microscope slide and cover glass with 70% ethanol and Kimwipes to remove any debris.

Problem 4

Fast photobleaching under fluorescence microscope. (Step 9).

Potential solution

Always focus with the DIC channel to indicate where the cells are and in focus, then only proceed to capture both DIC and mCherry channel using the automated function. This will minimize the exposure of cells to the illumination light.

Problem 5

Too few cells observed in a single view under microscope. (Step 10).

Potential solution

More images must be taken if the resuspended cells are in low concentration. To minimize the number of images needed to be taken to achieve N = 200 cells, concentrate by centrifuge the resuspended cells in 2400 × g 1 min, and remove some supernatant (PBS) before resuspending and view under microscope again.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Ee Sin Chen (bchces@nus.edu.sg).

Technical contact

Questions about the technical specifics of performing the protocol should be directed to and will be answered by the technical contact, Kim Kiat Lim (bchlimk@nus.edu.sg).

Materials availability

This study did not generate new unique reagents.

Data and code availability

The published article includes all [datasets/code] generated or analyzed during this study.