Stevej:Cell counting/hemocytometer

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Contents

Overview

Counting cells with a hemocytometer.

Materials

  • Tube of cell suspension
  • Hemocytometer
  • Trypan Blue 0.4%


Procedure

  1. Combine 20μl cell suspension with 20μl of Trypan Blue. Mix.
  2. Load 10μl into each of the 2 hemocytometer "sides" using the capillary action created by the coverslip and the hemocytometer. Avoid bubbles.
  3. Count the number of cells in 4 corners of the gridded area of each side. (See below) White cells are live and blue cells are dead/dying.
  4. Record cell count and repeat for the other side of the hemocytometer.
  5. If the two cell counts are roughly equivalent, average the cell counts. Otherwise, recount.
  6. Mean cell count/4 * Dilution = Cells x 104/ml.
  7. Above example, Mean cell count/4 * 2 = Cells x 104/ml.
demo

Notes

  1. Average number of 3T12 cells on a confluent:
    1. T175: 35-50M (LSJ, N=2)
    2. 10cm dish: 3M (VT)
  1. Cell doubling time:
    1. 3T12: ~24hrs.
    2. RAW:
    3. BMM:

References

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