# Introductionth to Cell and Tissue Culture - Jennie P.

ISBN 0-306-45859-4

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Figure 5.3.

The Coulter particle counter can be used for cell number determination. With this model counter, data collection and coincidence correction are automatically stored in the computer at right. The data can be easily transferred to a spread sheet for plotting.

count, which is then displayed on the LED readout. The counts shown include both viable and nonviable but intact cells. The counting threshold can be set to avoid counting cell debris. Cells must be a singlecell suspension to obtain accurate cell counts with these machines. The machines will also underestimate cell number in a manner related to the cell density. As the density becomes greater, there is an increasing chance that two cells will pass through the aperture so close together that they will only be counted as a single cell. This error can be corrected for by a mathematical formula that is used to generate the "coincidence correction tables" provided with the machines. This can be used to correct the counts manually in older model Coulter counters and is done automatically in the newer model counters.

Materials

1. Trypsinized cell suspension

2. Counting cuvette (blood dilution vials, Scientific Products)

3. Isoton solution

Procedure

1. Allow the counter to warm up prior to trypsinization.

2. Trypsinize cells as above.

3. Resuspend the cells in a total of 1 ml PBS and transfer to a cuvette. If this is a routine count for subculturing, remove 200 ^l from the neutralized cell suspension and add it to a cuvette.

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4. QS to 10 ml with Isoton solution.

5. Place the cuvette on the aperture platform and carefully immerse the aperture tube into the cuvette, taking care that the electrode is also immersed.

6. Open the stopcock (turn 90ja to the vertical) on the aperture tube until the display resets to zero.

7. Close the valve.

8. The counter will sample 0.5 ml of the cuvette suspension.

9. Reset the counter and take a second count. This is important because in older machines particularly the polarity can affect the count itself; so, for complete accuracy, one should make two counts/cuvette.

10. If the count is greater than 10,000, check the number against the coincidence correction chart supplied by Coulter for this purpose. The machine is quite accurate between 10,000 and 100,000 cells/0.5 ml. If the count exceeds 100,000, dilute the cell suspension 1:2 or 1:4 and recount in the accurate range.

11. Calculate the cell number by multiplying the average of the two displays by 20 (volume of 10 ml), which gives the total number in the cuvette. This will also give the total number in the 1-ml sample. In the case where 200 ^l is the sample volume, multiply by 5 to get the number of cells/ml. A shortcut when using 200 ^l as a sample volume is to add two zeros to the readout on the display to get a direct number of cells/ml; that is, 51,234 will be read as 5,123,400 or 5.1 x 106 cells/ml. (Note: This number must be corrected for coincidence for an accurate count.) This is only accurate when a count of greater than 10,000 is displayed. If the cells had to be further diluted to get within the range of the machine, multiply the calculated cell number by this factor. Note: Mastering the calculations involved in determining total cell number from counter or hemocytometer raw counts is frequently the most difficult task for a beginning student to learn.

If the equipment is in working order and being used correctly, all means of obtaining total cell number, including the hemacytometer, Coulter Counter, and the more sophisticated FACS machines, should be in good agreement. As always, each has different potential sources of error. Not having a single cell suspension is the most frequent source of error for cell counts. Allowing the suspended cells to sit in PBS or another buffer for prolonged periods can also lead to cell death and, with fragile cells, the fragmentation of dead cells and an underestimate of the number of cells.

Generating a Growth Curve

Materials

1. Cell cultures

2. Ten 60-mm tissue culture plates

3. Growth medium, 55 ml

4. Trypsin'CEDTA solution

5. Soybean trypsin inhibitor (1 mg/ml stock), if serum free Procedure

1. Trypsinize cells as previously described.

2. Resuspend cells in a total of 10 ml medium; this can be serum-containing medium, or if serum free, add 1 ml of soybean trypsin inhibitor.

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3. Wash by centrifugation in a clinical centrifuge, 900 rpm 3"C4 min

4. Resuspend the pellet in 5 ml of medium; remove an aliquot for counting.

5. Dilute an appropriate amount of the cell suspension into 55 ml of growth medium so that the total cell number in the tube is 5.5 x 105.

6. Resuspend the cells well and add 5 ml to each plate. This should result in a seeding density of 5 x 104 cells/plate (2 x 10'C3 cells/cm2).

7. Count the remaining 5 ml of cell suspension to establish the actual seeding density. Note: It is wise to always count an aliquot of cells at the beginning and end of setting up any experiment. The counts should be identical. If these vary by more than 10%, there is a technical problem that should be corrected. If the variance is too great, the experiment should be discarded, since all plates will not have the same inoculum density.

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