Mar 20, 2018

Public workspaceCounting Cells with Hemocytometer

DOI
dx.doi.org/10.17504/protocols.io.nxsdfne
  • Tyler C. Moore1
  • 1Bellevue University
  • Bellevue University Natural Sciences Laboratories
Tyler Moore
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DOI: dx.doi.org/10.17504/protocols.io.nxsdfne
Protocol Citation: Tyler C. Moore 2018. Counting Cells with Hemocytometer. protocols.io https://dx.doi.org/10.17504/protocols.io.nxsdfne
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol in our group and it is working.
Created: March 20, 2018
Last Modified: March 28, 2018
Protocol Integer ID: 10962
Abstract
Counting cells with a hemocytometer is an easy way to determine relatively accurate numbers of viable cells.  After determining cell counts, cells can be passaged, frozen away, or used for an experiment at a particular density.  
Guidelines
Keep cells sterile by wearing gloves, ethanol treating your hands and surfaces, using sterile pipette tips, and only opening vials with cells in the BSL-2 laminar flow hood.
Materials
MATERIALS
ReagentTrypan Blue 100 mL STEMCELL Technologies Inc.Catalog #7050
ReagentComplete DMEM (DMEM 10% HI FBS 50ug/mL Gentamycin)Gibco - Thermo Fisher Scientific
ReagentHemocytometer (Neubauer)
ReagentCompound Microscope
Collect cell suspension
Cell suspension must be in a known volume.  Adherent cells can be detached by methods appropriate for your particular cell line.
Dilute an aliquot of cells in Trypan Blue
Use a 1:10 dilution for many cells, 1:2 dilution if working with fewer cells
Need a total volume of 10ul; Can do 10ul of cell suspension into 90ul Trypan Blue (1:10) to ensure plenty of excess cells and limit pipetting errors associated with pipetting volumes less than 10 ul
Add cells to hemocytometer
Using a P10 micropipette, transfer 10ul of cell suspension + Trypan Blue under the cover slip on a hemocytometer
Count Cells
Observe the hemocytometer under the compound microscope
Count the cells within the squares
Count cells on 2 of the 4 perimeter lines
Only count live (non-blue) cells
Valid counts should include at least 50 cells


Calculate cell concentration
Cells/mL = (Cell count)/(number of chambers counted) x dilution x 104
EX: If you diluted your cells 1:10 in trypan blue, counted chambers 1 and 4, and counted a total of 150 cells:150/2 = 75 x 10 x 104 = 7.5 x 106 cells/mL
NOTE: Concentration (cells/mL) x total volume (mL) = total number of cells