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Ch 8.Immunohistoch / immunology
Ch 10.GC/MS, NMR and Proteomics
Protocols / Cellular Biology / Tissue Culture Technques 1, 2, 3, 4.
Tissue Culture Technique 4
6. Freezing cells.
a.
Harvest cells as usual and wash once with complete medium.
b. Resuspend cells in complete medium and determine cell count/viability.
c.
Centrifuge and resuspend in ice-cold freezing medium: 90% calf serum/10% DMSO, at 106 - 107 cells/ml. Keep cells on ice.
d. Transfer
1 ml aliquots to freezer vials on ice.
e. Place in a Mr. Frosty container that is at room temperature and that has sufficient isopropanol.
f.
Place the Mr. Frosty in the -70C freezer overnight. Note: Cells should be exposed to freezing medium for as little time as possible
prior to freezing
g Next day, transfer to liquid nitrogen (DON'T FORGET) and log in the "Liquid Nitrogen Freezer Log" Book.
7. Viable
cell counts. USING A HEMOCYTOMETER TO DETERMINE TOTAL CELL COUNTS AND VIABLE CELL NUMBERS (Reference: Sigma catalogue) Trypan blue
is one of several stains recommended for use in dye exclusion procedures for viable cell counting. This method is based on the principle
that live cells do not take up certain dyes, whereas dead cells do.
1. Prepare a cell suspension, either directly from a cell culture
or from a concentrated or diluted suspension (depending on the cell density) and combine 20 ul of cells with 20 ul of trypan blue
suspension (0.4%). Mix thoroughly and allow to stand for 5-15 minutes.
2. With the cover slip in place, transfer a small amount of
trypan blue-cell suspension to both chambers of the hemocytometer by carefully touching the edge of the cover slip with the pipette
tip and allowing each chamber to fill by capillary action. Do not overfill or underfill the chambers.3. Starting with 1 chamber of
the hemocytometer, count all the cells in the 1 mm center square and four 1 mm corner square. Keep a separate count of viable
and non-viable cells.4. If there are too many or too few cells to count, repeat the procedure either concentrating or diluting the
original suspension as appropriate.5. The circle indicates the approximate area covered at 100X microscope magnification (10X ocular
and 10X objective). Include cells on top and left touching middle line. Do not count cells touching middle line at bottom and right.
Count 4 corner squares and middle square in both chambers and calculate the average.6. Each large square of the hemocytometer, with
cover-slip in place, represents a total volume of 0.1 mm3 or 10-4 cm3. Since 1 cm3 is equivalent to approximately 1 ml, the total
number of cells per ml will be determined using the following calculations:Cells/ml = average cell count per square x dilution factor
x 104;
Total cells = cells/ml x the original volume of fluid from which the cell sample was removed; % Cell viability = total viable
cells (unstained)/total cells x 100.