Sec: Name: Pre-Lab 4 Worksheet Students will be counting cells using an automated the Countess. has traditionally been accomplished using a Hem Trypan Blue Assay. Learning and understanding his type of calculation also informs you of how the automated cell counter owever, determining total cell number and cell viability ocytometer and the Countess) determines the final numbers for your experiments Hemocytometers have a grid to help you not lose your place as you count the cells. There are nine ‘”big squares’, each 1 mm x 1 mm (see image to the right). To begin, count all the cells in the middle square and the four corner squares (ie. A B, C, D, and E in the diagram). Each ‘big square” has a surface area of 1 mm x 1 mm, plus it is 0.1 mm deep. 1 mm 1 mm Therefore, the volume of liquid that covers that big square (0.1 mm3) is 0.0001ml or 1.0 x104 ml because there are 1000 mm3 in 1 ml. Apoptotic (dying) and dead cells are more permeable than those of healthy cells Trypan blue can cross the plasma membrane of these apoptotic and dead cells. Therefore, cells that turn blue after the trypan blue assay are considered unhealthy or nonviable. Once the cell pellet has been re-suspended, we typically remove 100ul of cell suspension and place it into a microcentrifuge tube. Then we add 100ul of trypan blue to the cel suspension for a 1:1 dilution of the cells. This is a dilution factor of 2. We can use the trypan blue exclusion assay to determine cells per mL, total cells in the suspension, percent viable and percent nonviable cells. hemesa Use the equations below in the practice problems the equation to calculate total cells per ml Solve for X (Cells per mL)- 1) a. x 1×10 x 2 (dilution factor of Trypan Blue) number of big squares 2) the equation to calculate total cells in cell suspension (in the tube) a. Solve for X (Total cells in cell suspension) cells per mL x total volume of cell suspension 3) the equation to calculate percent viable cells a. Solve for x (percent viable) viable cells countedx 100 total cells counted (viable + nonviable) 4) the equation to calculate percent nonviable cells a. Solve for x (percent nonviable) x 100 total cells counted (viable+ nonviable) Questions about cell counting A. What if a cell is on the lines that define a “big square”? Do you count it? The general rule of thumb is that ifmore than 50% of the cell is iside of the big square most important thing is to be consistent. For example, if you and your group decide that in order for counted, it should be completely inside the first big square, make thing for the other four big squares you should count it. The sure you and your group members do the same B. What if you see a large “clump of cells? How do you count i? PIK2 cells produce a lot of extra cellular matrix and sure your cell pellet is completely re-suspended before cou your cell pellet, try your best to estimate the number of cells in a down more in future experiments. can clump together. This is why it is very important to make ed in solution before counting the cells. If you did not fully re-suspend clump and make sure to pipet the cells up and Practice Problem 1- Counting Cells: Using the equations described on the previous page to determine the concentration of cells (cells/ml), total cells in cell suspension, percent viable cells and percent nonviable cells The following hemocytometer grid shows PtK2 cells that were re-suspended in 4mL of media. Solve for the four equations above (C-F) and SHOW YOUR WORK FOR EACH o Which cells are represented by this open circle? B. Which cells are represented by this dark circle? C. Total cells/mL 0 D. Total cells in suspension . E. Percent Viable Cells- 0 F. Percent Non-viable cells-