How to Count Fresh Primary Cells

When you receive a primary cell product for your research needs, it is recommended that you count the number cells to ensure you received the proper order. It is important to do this immediately after you receive the product, as most primary cell sources are not liable for any cell loss during subsequent processing.

How to Count

In a biosafety hood, use a 5 mL pipet to mix the cell suspension very well and to separate any cell clumps. For any cells in a 1.5 or 2mL tube, mix cells with a P1000 pipet.

Take a 10 μL sample from the vial. Mix the 10μL sample with 10μL of trypan blue. Dilute the cells if necessary and count the number of cells in 4 quadrants of hemacytometer to determine the viability.

Common Dilution Factors:

Tube Size: 1.5 to 2mL, Cell Number/Unite Number: 0.5 to 1 x 10 to the 5th power, Cell Sample: 10μL, 0.4% Trypan Blue Amount: 10μL, PBS (1x): ---, Dilution Factor: 2

Tube Size: 5 mL, Cell Number/Unite Number: 5 x 10 to the 6th power, Cell Sample: 10μL, 0.4% Trypan Blue Amount: 10μL, PBS (1x): ---, Dilution Factor: 2

Tube Size: 15 mL, Cell Number/Unite Number: 10 x 10 to the 6th power, Cell Sample: 10μL, 0.4% Trypan Blue Amount: 10μL, PBS (1x): ---, Dilution Factor: 2

Tube Size: 15 mL, Cell Number/Unite Number: 15 to 20 x 10 to the 6th power, Cell Sample: 10μL, 0.4% Trypan Blue Amount: 10μL, PBS (1x): 80 μL, Dilution Factor: 10

Tube Size: 50 mL, Cell Number/Unite Number: 30 to 100 x 10 to the 6th power, Cell Sample: 10μL, 0.4% Trypan Blue Amount: 10μL, PBS (1x): 80 μL, Dilution Factor: 10

N = # of cells counted on all 4 quadrants of a hemacytometer

d = dilution factor

Trypan Blue Method

Equation for Cell Count:

# of cells/vial = N / 4 x d x ____mL x 104

Equation for Viability:

# of cells excluded by trypan blue / total number of cells x 100% = ______%

Table 2: Common Dilution Factors for MNC Products with RBC (Cord Blood and Bone Marrow Mononuclear Cell Products)

Tube Size: 50 mL, Cell Number/Unite Number: 100-200 x 10 to the 6th power, Cell Sample: 25μL, *3% Acetic Acid with Methy-lene Blue Solution: 475μL

Tube Size: 50 mL, Cell Number/Unite Number: 200-400 x 10 to the 6th power, Cell Sample: 25μL, *3% Acetic Acid with Methy-lene Blue Solution: 475μL

Methylene Blue Method

Equation for Cell Count:

# of cells/vial_____ /4 x 20 x d x ___mL x 106

NOTE: Methylene Blue Method will stain DNA to quantify the nucleated cells in a sample. Methylene Blue will not give you the cell viability. To find cell viability use Trypan Blue Method.

Troubleshooting:

If your cell count is significantly lower than what the supplier claims, taking the following steps is advised:

1. Check the cell suspension and see if there are any cell clumps. If so, try to separate the cells using a P1000 pipet tip. Most of the cell clumps should be small and easily separable.

2. The optimal cell concentration is 50 to 100 cells on one large hemacytometer square (total 200 to 400 per hemocytometer). Please make the proper dilution or adjust the concentration and count again.

3. Immediately report any significant discrepancies to your cell supplier.

To clean the counting chamber:

After counting, remove the cover glass and clean the counting chamber with water or a mild cleaning solution (10% solution of bleach). Rinse counting chamber with 70% ethanol and dry the counting chamber with a soft cloth or wipe.


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http://www.AllCells.com/ is a global biotechnology company founded in 1998 to provide researchers with a reliable supply of biologically relevant, consistent quality primary cells that enable the advancement of scientific research in the areas of cell biology, oncology, virology, hematology, infectious disease and stem cell research.


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