Why is trypan blue used when counting cells on a hemocytometer?

Trypan Blue is one of several stains recommended for use in dye exclusion procedures for viable cell counting. This method is based on the principle that live (viable) cells do not take up certain dyes, whereas dead (non-viable) cells do. Staining facilitates the visualization of cell morphology.

How do you calculate the dilution factor of a Hemocytometer?

Dilution Factor = Total Volume (Volume of sample + Volume of diluting liquid) / Volume of sample. Total viable cells/Sample = Viable Cells/ml x The original volume of fluid from which the cell sample was removed. Volume of media needed = (Number of cells needed/Total number of viable cells) x 1000.

How do you dilute trypan blue?

Mix 1 part of 0.4% trypan blue and 1 part cell suspension (dilution of cells). Allow mixture to incubate appoximately 3 minutes at room temperature. Note: Cells should be counted within 3-5 minutes of mixing with trypan blue, as longer incubation periods will lead to cell death and reduced viability counts.

When to add trypan blue to hemocytometer?

3. Mix thoroughly and allow to stand 5 to 15 minutes. Note: If cells are exposed to trypan blue for extended periods of time, viable cells may begin to take up dye as well as non-viable cells, thus, try to do cell counts within one hour after dye solution is added.

What is the dilution factor for hemocytometer cell counting?

1. Transfer 200 µl of the cell suspension into a 1.5 ml microfuge tube. 2. Add 300 µl of PBS and 500 µl of 0.4% trypan blue solution to the cell suspension (creating a dilution factor of 5) in the centrifuge tube. 3. Mix thoroughly and allow to stand 5 to 15 minutes.

How to count cell suspension in a hemocytometer?

Mix the cell suspension and add 20 µL to the tube or well containing 3% Acetic Acid with Methylene Blue. Ensure that the cell suspension to be counted is completely resuspended. Before the cells settle, place a suitable volume of a cell suspension (20 – 200 μL) in a centrifuge tube.

When to use more or less PBS in hemocytometer?

If there are more than 200 cells per well, dilute your suspension with PBS by an appropriate volume. If the cell density per large square is less than 50 then go back to your culture and use more cells and less PBS.