Determining the number of cells and culture is important in the standardization of culture conditions and when performing experiments where precise cell numbers are needed. Here we will show you how to count cells using a hemo cytometer, and calculate the concentration of cells present in suspension. We will also show you how to determine the number of viable cells present in your culture with the Trian Blue Viability Assay.
Hi, I'm Ricky here at the Dr.LAN's Laboratory at Molecular Pathology Laboratories Network in East Tennessee. Today I'm going to show you how to count cells and suspension and how to determine their viability. In order to prepare the hemo cytometer for cell counting, you first want to clean its surface and the accompanying cover slip with 70%ethanol.
The cover slip and slide should be clean, dry, and free from lint, fingerprints and watermarks. Next, wet the edges of the hemo cytometer slightly with tap water and press the cover slip down over top of it. The cover slip should rest evenly over the silver counting area.
Now we are Ready for the cell suspension to prepare the cell suspension For counting. First, if the cells are grown in monolayer cultures, detach the cells from the surface of the plate using trypsin as seen in the video for pasting cells. Once the cells have detached from the plate, dilute them as needed to obtain a uniform suspension.
Be sure to disperse any clumps in the suspension when using the hemo cytometer. A maximum cell count of 20 to 50 cells per One millimeter square is recommended. When you're ready to load the hemo Cytometer with your cell sample, use a micro pipetter to transfer the cell suspension to the edge of the hemo cytometer counting chamber.
Hold the pipette tip under the cover slip and dispense one drop of suspension. The suspension will be drawn under the cover slip by capillary action. After you have filled the first chamber, fill the second counting Chamber and you are ready to count your cells before you begin to count.
The cells allow the cells To settle for a few minutes. It also helps to blot off any excess liquid once the cells have settled. You can view the slide on the microscope with a hundred x magnification.
You should see the large central area of the grid, which is bordered by a set of three parallel lines. Subdivisions within the large central area are also bordered by three parallel lines and each subdivision is divided into 16 smaller squares by single lines. Cells within this area should be evenly distributed without clumping.
If cells are not evenly distributed, wash and reload. The hemo cytometer use a handheld counter to count cells in each of the four corner and central squares count cells. Touching the middle line of the triple line on the top and left of the squares did not count cells touching the middle line of the triple lines on the bottom or right side of the square.
Repeat counts for the other counting chamber. Once you have counted the cells, you can determine the number of cells per milliliter. To do this, take the average count of cells per square and multiply by the dilution factor and by 10, 000 to get the number of cells in your sample.
Multiply the cells per milliliter with the total volume of the cell suspension from which the sample was taken. Now we are ready to determine the Viability of our suspension. To determine the number of viable Cells in your sample, we will use the D trian blue first combine 0.5 milliliters of 4%trian blue 0.03 mils, HBSS and 0.1 mils of the cell suspension in a small tube mixed thoroughly, and let the tube stand for five minutes before loading the hemo cytometer.
The viable cells should be unstained, but dead cells will appear blue under the hemo cytometer. Count the total number of cells and the total number of viable cells, unstained cells, and calculate the percent viable cells in your sample. Once you are finished, decontaminate the cover slip in hemo cytometer by rinsing with 70%ethanol and then deionized water.
Let it air dry and store It until future use. We've just shown you how to determine the number and viability of your cultured cells. So that's it.
Thanks for watching and good luck with all your experiments.
