TITLE: Manual Cell Counting 
AUTHORS: Aparna Kumar, Dana AL-Azzeh, Schuele Lab

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Manual counting of cells using a Hemocytometer.</div></div>

[STEPS]
?. [Preparation for Counting Cells]
Remove  of the solution containing the single cells using a p20 micropipette and add to a 1.5 ml microcentrifuge tube or onto a piece of parafilm.
10 µl
?. [Preparation for Counting Cells]
Centrifuge the Trypan Blue and remove  from the top of the Trypan Blue as not to get any debris using a p20 micropipette.
10 µl
?. [Preparation for Counting Cells]
Add the  of Trypan Blue to the 1.5 ml microcentrifuge tube or the parafilm conataining the cells and gently mix by pipetting up and down.
10 µl
?. [Preparation for Counting Cells]
Wipe the hemocytometer and a matching coverslip with 70% ethanol and place in the biosafety cabinet.
?. [Preparation for Counting Cells]
Place the coverslip onto the center of the hemocytometer.
?. [Preparation for Counting Cells]
Remove  of solution from the 1.5 ml microcentrifuge tube or the parafilm using a p20 micropipette.
10 µl
?. [Preparation for Counting Cells]
Place the tip of the pipette at the bottom edge of the coverslip and slowly pipette the solution into the hemocytometer. The bottom chamber should be filled.
?. [Preparation for Counting Cells]
Remove another  of solution and add to the top chamber by placing the tip of the pipette at the top edge of the coverslip and slowly pipetting the solution into the top chamber.
10 µl
?. [Preparation for Counting Cells]
Place the filled hemocytometer on a microscope and focus the microscope such that the cells and the grid lines in each chamber are clearly visible.
?. [Counting the Cells]
Pick one of the 9 boxes in the top grid (the grid in the top chamber) to begin counting. Note: Cells stained blue are dead cells and should only be counted to determine the viability, not the cell count.
?. [Counting the Cells]
Any cells that are on the bottom and right edge lines should be counted while any cells overlapping the top and left edge should not be counted.
?. [Counting the Cells]
Using a tally counter, keep track of the number of cells counted in one box.
?. [Counting the Cells]
Note the total cells in one box and reset the counter.
?. [Counting the Cells]
Repeat steps 10 - 13 four more times, choosing a different box in the same grid.
?. [Counting the Cells]
Move on to the bottome grid (the grid in the bottom chamber) and repeat the process of counting by counting the cells in 5 boxes and making a note of the counts.
?. [Calculating the Number of Cells]
Find the average number of cells per box by adding up the total number of cells and dividing by the number of boxes that where counted. Note: In this protocol, 10 boxes where counted total, 5 in the top grid and 5 in the bottom grid, so the total number of cells should be divided by 10.
?. [Calculating the Number of Cells]
Multiply the final number from step 16 by 2 and then by 10,000. The result is the total number cells in the original solution (before the addition of Trypan Blue) per mL.
?. [Calculating the Number of Cells]
Total number of cells = (number of cells per mL) * (mL of original solution containing the cells)
?. [Calculating the Number of Cells]
Cell viability = 100 * (Live cells)/(Live cells +Dead cells)