FibroblastCellSystems5

Hemacytometer Reference Figure 1

5.Determine the Cell Count.

a. Calculate the total cells counted in the four corner squares.

1) If the total cell count is less than 100, or if more than 10% of the cells counted appear to be clustered, carefully re-mix the original cell suspension and repeat steps 2 through 4 (above).

2) If the total cell count is greater than 400, dilute the suspension so the count will be 100-400 cells. Then repeat Steps 2 -4 (above).

NOTE: If satisfactory results are not achieved, contact your Clonetics? Technical Specialist by telephoning 800-852-5663.

b. Calculate the cell count using the equation: cells/ml = (n) x 104,

where: n = the average cell count per square of the four corner squares counted.

Example:If the calculated average (n) of cells in the four 1 mm corner squares of the hemacytometer is 30:

cells/ml = (n) x 104 (or) cells/ml = 30 x 10,000 = 300,000 cells/ml.

c. Determine the total number of cells in the total suspension volume.

1) Determine the total volume of the cell suspension.

2) Multiply the volume of the cell suspension by the "cells/ ml" value calculated above.

Example:If the initial suspension volume is 2 ml:

cells/ml x total volume = 300,000 cells/ml x 2 ml = 600,000 cells.

APPENDIX C 
ASSESSMENT OF CELL VIABILITY WITH TRYPAN BLUE

Background

Trypan blue is a dye that enables easy identification of dead cells. Dead cells take up the dye and appear blue with uneven cell membranes. By contrast, living cells repel the dye and appear refractile and colorless.

Using Trypan Blue

1. Prepare the hemacytometer for use.

a. Carefully clean all surfaces of the hemacytometer and cover slip.

b. Take care to ensure that all surfaces are completely dry using non-linting tissue.

c. Center the cover slip on the hemacytometer.

2. Transfer 50 ml of 0.4% Trypan Blue into a clean tube.

3. Add 50 ml of the prepared cell suspension into the tube containing the stain.

4. Mix the solution thoroughly, but gently. Take care to avoid making excessive bubbles.

5. Allow the mixture to sit for 2-3 minutes after mixing. (Do not let the cells sit in the dye for more than five minutes because both the living and dead cells will begin to take-up the dye after five minutes.)

6. Pipet approximately 9 microliters of the Trypan Blue/cell suspension mixture (this volume will vary with brand of hemacytometer) into one of the two counting chambers.

a. Use a clean pipet tip.

b. Be sure that the suspension is mixed thoroughly but gently before drawing the samples.

c. Fill the chambers slowly and steadily.

d. Avoid injecting bubbles into the chambers.

e. Do not overfill or underfill the chambers.

7. Determine Cell Viability.

a. Allow the suspension to settle in the chambers for at least 10 seconds.

b. Count all of the stained cells in each of the four corner squares of the hemacytometer.

c. Separately count all of the unstained cells in the same squares.

d. Calculate the cell viability using the equation:

% Cell Viability = number of unstained (living) cells / Total cells counted (stained + unstained) x 100%

Example: If a total of 300 cells (stained + unstained) are counted and 200 are identified as living cells (unstained), then the viability is calculated as:

% Cell viability =200 / 300 x 100% = 67%

IMPROVING CELL YIELD AND VIABILITY

Background

Several factors, or a combination of factors, contribute to low cell count and low cell viability. If cell yield or viability is unsatisfactory, use the following information to increase the success rate of future cultures.

Improving Cell Yield

If your cell yield is low (less than 50%), determine the cause(s) and possible solution(s) using the table below. Then subculture one more flask applying the appropriate solution(s).

Improving Cell Viability

If your cell viability is low (less than 50%), determine the possible cause(s) and solution(s) using the table below. Then subculture one more flask applying the appropriate solution(s).

Once you have determined how to achieve high yield and high viability, subculture the remaining flasks.