While counting cells with hemocytometer trypan blue exclusion should I calculate both the chamber (two counting grids) 5 square in each chamber? or only one chamber?
Each square occupiers 10^-4 ml or cc so avg no of cells * dilution factor * 10,000.
Why is it multiplied with 10,000?
I used this methos to count HeLa (cervical cancer) and F0 (Myeloma cells) cells.
1. For "X" cells, take sample after harvesting the cells,
2. Take 10ul trypan blue in a microtube+ 10ul cell sample…..there should not be any bubbles.
3. Mix it well without any bubbles.
4. Take 10ul to the hemocytometer carefully.
5. Observe it under microscope.
6. Count the cells over 4 squares from 4 corners.
Finally count it,
Number of cells from 4 squares/4 = AVG of cell number
AVG of cell number X 2 / 10^(-4) = No. of cells in 1 ml
Here,
1 square contains 10^-4ml of sample. That's why it is divided by 10^-4.
2 is dilution factor {total vol (10ul sample+ 10ul trypsin blue)/ vol of sample (10ul)}
Multiply with volume of the test tube. Like if there was 10ml in the test tube, multiply the result with 10 to get the cell number in the test tube.
As each of the “large” squares is 1mm x 1mm, and the area
between the slide and the coverslip is 0.1mm, then the volume above
each “large” square is 0.1 mm3= 0.0001 ml
To give you cell count per ml, you need multiply to 10000.
You can think of each large square as having a volume of 100nL. So you have to multiply by 10,000 in order to convert the number of cells in 100nL to the number of cells per mL.
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