Appendix:

Calculations

The formula used to make our various dilutions was:

M1xV1 = M2xV2

M1 = stock concentration

M2 = final concentration

V1 = how much of the stock we need (the volume)

V2 = the volume of the treated cell culture well

For example: How do we make a working stock of 600 nM E2 (Estradiol)? We need to make 2 mL of the drug.

(10uM) (x) = (600nM) (2000uL)

(10uM) (x) = 12 x 10^6nM x uL

x = (1.2 x 10^6nM x uL) / 10000nM

x = 120 uL

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To count the cells a hemocytometer was used. This is a glass slide with a chamber for counting cells within a given volume. The counting is done visually using a microscope, and four counts are done for each sample, or different treatment. These counts are then averaged, and the total number of cells is determined using the following formula:

Cells/mL = the average count x 10^4

Total Number of Cells = cells/mL x original volume from which sample was taken