A Simple Method for in situ Quantification of Cells on Carriers

Materials and Reagents

1. 1.5 ml Eppendorf tubes (Sarstedt, catalog number: 72.692.005)

2. Pipette tips (Filtered) (Thermo, catalog numbers: TF112-1000-Q [1,000 µl], T104RS-Q [10 µl], TF140-200-Q [200 µl])

3. 24-well clear plates (Corning, catalog number: 3524-ND)

4. 75 cm² T-flasks (Grenier, catalog number: 658-975)

5. 500 ml vacuum filter bottle system, Sterile (Corning, catalog number: 431097)

6. Counting vessels (Invitrogen, catalog number: C10283)

7. Aluminum foil (Nidix)

8. Vero cells (WHO, catalog number: RCB 10-87)

   Note: Cells used for the calibration curve must be the same as cells on carriers.

9. L-alanine-L-glutamine (Biological Industries, catalog number: 03-022-1B)

10. NutriVero FLEX-20 (Biological Industries, catalog number: 05-069-1A)

11. Pen-strep antibiotics (Biological Industries, catalog number: 03-031-1C)

12. Phosphate buffered saline (PBS) (Biological Industries, catalog number: 02-020-1A)

13. Recombinant trypsin-EDTA solution (Biological Industries, catalog number: 03-079-1A)

14. Trypan blue solution (Biological Industries, catalog number: 03-102-1B)

15. Alamar blue (Promega, catalog number: G808)

16. Cell medium (see Recipes)

Equipment

1. Sterile tweezers (United Scientific Supplies, catalog number: 20A00R146)

2. Sterile scalpel (Asp medical, catalog number: LDT455)

3. Pipettors 200 and 1,000 µl (ThermoFisher Scientific, catalog numbers: 4652140, 4652080)

4. Incubator at 37°C with 5% CO₂ (ThermoFisher Scientific, model: Heracell 150i)

5. Laminar flow hood (ThermoFisher Scientific, model: Msc-advantage)

6. Refrigerator 4°C (Mondial Elite, model: BEV PV 40 MED)

7. Centrifuge (Eppendorf, model: Minispin plus spin)

8. SPARK plate reader (NEOTEC BIO, TECAN)

9. Cytometer cell countess II (Invitrogen, model: AMQAX1000)

10. Heater (Talboys, microplate heater)

Software

1. Microsoft Excel 2010

Procedure

1. Preparing the cells

   1. Maintain the cells in cell medium (see Recipes) in 75 cm² T-flasks at 37°C under 5% CO₂.

   2. Harvest the cells by washing the flask one time at RT with 5 ml of PBS at room temperature (RT), followed by incubation at 37°C with 2 ml of trypsin-EDTA for 5 min.

   3. Transfer the cells from the flask to a 1.5 ml Eppendorf tube.

   4. Centrifuge the cells at RT for 5 min at 1,200 × g.

   5. Remove the supernatant with a 1 ml pipette.

   6. Resuspend the pellet in 1 ml of cell medium at 37°C, by pipetting the solution up and down five times.

   7. Take a sample of 50 μl from the cell solution, and mix 1:1 with trypan blue in a 1.5 ml Eppendorf tube.

   8. Count the cells with a cytometer cell countess.

   9. Dilute as necessary by adding cell medium to reach a concentration of 800,000 cells/ml (stock for calibration curve). A total of 2 ml is necessary.

   10. If the cells to be quantified were cultured and grew in different media than the one described here, the exact same conditions should apply to cells for the calibration curve.

       
       

2. Preparing the calibration curve

   1. Add 1 ml of fresh medium to wells 1A, 2A, 3A, 4A, and 5A of a 24-well plate.

   2. Place 2 ml of 800,000 cells/ml (stock for calibration curve, see A9) in well 6A.

   3. Take 1 ml from well 6A, and transfer it to the adjacent well (5A).

   4. Mix by gently pipetting five times with a 1 ml pipette.

   5. Take 1 ml from well 5A, and transfer it to the adjacent well (4A).

   6. Mix by gently pipetting five times with a 1 ml pipette.

   7. Take 1 ml from well 4A, and transfer it to the adjacent well (3A).

   8. Mix by gently pipetting five times with a 1 ml pipette.

   9. Take 1 ml from well 3A, and transfer it to the adjacent well (2A).

   10. Mix by gently pipetting five times with a 1 ml pipette.

   11. Take 1 ml from well 2A and dispose of it.

   12. Well 1A contains only 1 ml of fresh medium for negative control.

   13. Wells 2A-6A contain 50,000, 100,000, 200,000, 400,000, and 800,000 cells, respectively (Figure 1).

   14. Use another 24-well plate if there are more than 18 samples. In that case, prepare another calibration curve in the new plate as described above.

       
       

       
       Figure 1. Preparation of the calibration curve. The cell stock was brought to 800,000 cells/ml, and 2 ml of it were added to well 6A in a 24 well plate. In the same row, 1 ml of culture medium was placed in the other five wells (1A-5A). A serial dilution was performed by taking 1 ml of 800,000 cell/ml from well A6 to the adjacent well (5A), and serially across the entire row so that wells contained 800,000, 400,000, 200,000, 100,000, and 50,000 cells. The last well in the row (1A) was left with 1 ml fresh medium as a blank control.

       
       

3. Determining the number of cells on carrier

   1. Add 1 ml of cell medium to all empty wells in rows B, C, and D in the plate.

   2. Place one carrier in a well containing 1ml of fresh medium.

   3. Add 100 µl of Alamar blue to all wells in the plate.

   4. Cover the plate with aluminum foil (protect from light).

   5. Incubate the plate for 1 h at 37°C.

   6. Read the plate in the SPARK plate reader, with Ex 550 nm, Em 580 nm filters.

Data analysis

1. Transfer the data to an Excel file.

2. In the Excel file, write the number of cells that were used in the calibration curve (50,000-800,000) on column A, and include the blank (no cells, from well A1).

3. Insert the corresponding fluorescence intensity values in the adjacent column (B).

4. Subtract the value of the blank from all fluorescence values, and write the results in the adjacent column (C). see example below (Figure 2).

   
   

   
   Figure 2. Example of data layout for analysis. The number of cells that were used for the calibration curve and their fluorescence intensity values are in columns A and B in an excel file. The fluorescence of the blank is subtracted from all values and written in column C.

   
   

5. Write the fluorescence results from all other wells (containing cell-carriers). If several carriers were taken from the same vessel/reactor, write all results in the same column.

6. Average the results from step 5 (if more than one carrier from the same experiment) and subtract the value of the blank.

7. Calculate the number of cells on each carrier using the excel FORECAST formula based on the calibration curve (Figure 3).

   
   

   
   Figure 3. Example of data analysis. The fluorescence results from all wells (containing carriers) are written and their average is calculated. The amount of cells on the carriers is calculated by the FORECAST formula based on the calibration curve.

Notes

1. Every type of cells and carriers may be used.

2. Every validated 24-well plate may be used.

3. Every type of plate reader with suitable filters may be used.

4. For each 24-well plate, use its own calibration curve.

5. Cells for the calibration curve must be fresh and may be counted in every validated cytometer.

6. The method can quantify cells in different media, but the calibration curve must be performed under the same conditions.

Recipes

1. Cell medium

   Take 500 ml of FLEX-20 medium

   Add 5 ml of 200 mM L-alanine L-glutamine

   Add 0.5 ml of pen-strep antibiotics

   Filter the solution with a filter bottle

Acknowledgments

The protocol described here has been derived from the work of Rosen et al. (2021).

Competing interests

The authors declare not to have any competing interests

References

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