Stevej:Cell counting/hemocytometer: Difference between revisions
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#Combine 20μl cell suspension with 20μl of Trypan Blue. Mix. | #Combine 20μl cell suspension with 20μl of Trypan Blue. Mix. | ||
#Load 10μl into each of the 2 hemocytometer "sides" using the capillary action created by the coverslip and the hemocytometer. Avoid bubbles. | #Load 10μl into each of the 2 hemocytometer "sides" using the capillary action created by the coverslip and the hemocytometer. Avoid bubbles. | ||
#Count the number of cells in the gridded area of each side. (See below) White cells are live and blue cells are dead/dying. | #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. | ||
#Record cell count and repeat for the other side of the hemocytometer. | #Record cell count and repeat for the other side of the hemocytometer. | ||
#If the two cell counts are roughly equivalent, average the cell counts. Otherwise, recount. | #If the two cell counts are roughly equivalent, average the cell counts. Otherwise, recount. | ||
#Mean cell count/4 * Dilution = Cells x 10<sup>4</sup>/ml. | #Mean cell count/4 * Dilution = Cells x 10<sup>4</sup>/ml. | ||
#Above example, Mean cell count/4 * 2 = Cells x 10<sup>4</sup>/ml. | #Above example, Mean cell count/4 * 2 = Cells x 10<sup>4</sup>/ml. | ||
[[image:hemocytometer.png|none | [[image:hemocytometer.png|none|[[help:contents|demo]]]] | ||
==Notes== | ==Notes== | ||
#Average number of 3T12 cells on a confluent: | |||
##T175: 35-50M (LSJ, N=2) | |||
##10cm dish: 3M (VT) | |||
#Cell doubling time: | |||
##3T12: ~24hrs. | |||
##RAW: | |||
##BMM: | |||
==References== | ==References== | ||
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[[Category:Protocol]] | [[Category:Protocol]] | ||
[[Category:Needs attention]] <!--Delete this line once the protocol is complete--> | [[Category:Needs attention]]--> <!--Delete this line once the protocol is complete--> | ||
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Latest revision as of 08:51, 3 February 2009
Overview
Counting cells with a hemocytometer.
Materials
- Tube of cell suspension
- Hemocytometer
- Trypan Blue 0.4%
Procedure
- Combine 20μl cell suspension with 20μl of Trypan Blue. Mix.
- Load 10μl into each of the 2 hemocytometer "sides" using the capillary action created by the coverslip and the hemocytometer. Avoid bubbles.
- 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.
- Record cell count and repeat for the other side of the hemocytometer.
- If the two cell counts are roughly equivalent, average the cell counts. Otherwise, recount.
- Mean cell count/4 * Dilution = Cells x 104/ml.
- Above example, Mean cell count/4 * 2 = Cells x 104/ml.
Notes
- Average number of 3T12 cells on a confluent:
- T175: 35-50M (LSJ, N=2)
- 10cm dish: 3M (VT)
- Cell doubling time:
- 3T12: ~24hrs.
- RAW:
- BMM: