Splenocyte

Transcription

A hemocytometer is a device that is used for counting cells. It's a modified microscope slide, containing two identical wells, or chambers, into which a small volume of a cell suspension is pipetted. We have already removed 100 µL of our cell suspension and placed it in a micro-centrifuge tube. Dilute the suspension by adding 100 µL of Trypan blue. Trypan blue is a dye that helps us distinguish between living and dead cells. The dye passes through the membranes of dead cells so they will appear blue under a microscope. Living cells exclude and will appear mostly clear. Load both chambers by pipetting the suspension under the cover slip. Now place the hemocytometer under the microscope. Each chamber is divided into a grid pattern, consisting of 9 large squares. Each square has the same dimensions and contains 10 to the negative-fourth power mL of suspension. The rules for counting cells sometimes differ from lab-to-lab. In our lab, we count cells in the 4 large corner squares and the center square. Let's count the cells in the first square. One, Two, Three, Four, Five, Six, Seven, Eight... So what about the cells that are touching the outside boundaries of the square? In our lab we count the cells that touch the top and left boundaries, and we ignore the cells that touch the right and bottom boundaries. Nine. Ten. We need to count the number of both living and dead cells. Remember, the dead cells will appear blue. Occasionally you will see artifacts - objects or debris that appear blurry and don't have a well-defined shape. This is an example of an artifact. We won't include it in our count. Proper storage, cleaning, and handling of the hemocytometer will minimize the number of artifacts. There are 10 viable cells and 1 non-viable cell in the first square. Now, the top-right square. There are 9 viable cells and no non-viable cells. Next let's count the bottom-right square. There are 11 viable cells and no non-viable cells. And now the bottom-left square. There are 10 viable cells and 2 non-viable cells. And finally, let's count the cells in the center square. Sometimes cells will appear as clumps or small groups. It may be difficult to determine exactly how many cells are in a group. The method of counting clumps of cells differs from lab to lab, so be sure to follow the procedure in your lab. We will count this clump as 2 cells. There are 14 viable cells and no non-viable cells in the center square. The total number of viable, or living cells from all 5 squares is 54. The total number of non-viable cells is 3. Now that we have counted our cells, there are several calculations we need to perform. First, let's calculate the percentage of viable cells. Here's the formula. 54 viable cells, divided by 57Éthe total number of cellsâ gives us 0.947. Multiply by 100 and the percentage of viable cells is 94.7%. Next, let's determine the average number of cells-per-square. We counted 54 viable cells. We divide 54 by 5, because we counted in 5 squares. The average number of cells-per-square is 10.8 cells. Now let's calculate the dilution factor. The dilution equals the final volume divided by the volume of cells. Our final volume is 200 µL, because we started with 100 µL of cells and added another 100 µL of trypan blue. 200 divided by 100 is 2. Therefore the dilution factor is 2. Next we need to calculate the concentration of viable cells - the number of living cells/mL Our average count-per-square is 10.8. The dilution factor is 2. 10.8 times 2 times 10-to-the-fourth-power equals 216,000 cells/mL. We can write the concentration using scientific notation as 2.16 times 10-to-the-fifth power cells/mL. From our calculations, we now know the concentration of cells in our culture is 216,000 cells-per-milliliter and approximately 94.7 percent of those cells are viable, living cells.