Sasha Bakhru: Cell Culture Protocols
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| 6. Cell Culture Protocols (reference: Y.K. Chan)
6.1. Cell harvesting (1). Once you have obtained the petri dish/flask containing the fibroblasts, you would need to keep the lid closed until you have transferred the dish inside the cell culture hood. (2). Use a pipette to remove the media and dispose the media into a designated waste container. Wash the layer of cells with small amount of PBS (1-2mL) to remove residue serum that could retard trypsinization. (3). Remove the PBS and add 1 mL of trypsin solution (1X) over the layer of cells. Close the lid and incubate the cells in the incubator for approximately 5 minutes. You may look under the microscope to see the difference in the cell morphology as they have detached from the surface. Now most of the cells should be detached and if not, one can apply gentle shear forces by pipetting PBS solution over the cell layer surface. (4). Transfer the detached cells in the trypsin/PBS solution into a 15 mL centrifuge tube. Add the volume up to 5 mL with PBS. Then centrifuge at 1000 rpm for 2 minutes (note: make sure you check with the TA before centrifuging). After the cells have been centrifuged, one should be able to see a cell pellet at the bottom of the tube. Wipe the tube with ethanol before transferring back into the hood. (5). Open the tube under the hood and remove the supernatant. Now resuspend the clumped cells by pipetting with the DMEM media gently. Keep the pipette tip close to the bottom of the tube when resuspending the cells. Pipette for about 10 to 15 times and the cells should be uniformly resuspended. If clumps persist, ask the TA for assistance. 6.2. Cell counting using a hemocytometer (1). Clean the hemocytometer with RO water and then with alcohol. Make sure the device is dry from alcohol before applying the cell content. (2). The hemacytometer consists of two chambers, each of which is divided into nine 1.0 mm2 grids. A cover glass is supported 0.1 mm over these squares so that the total volume over each 1 mm2 square is 10-4 mL. The concentration per mL of cells = cells counted in each 1 × 1 mm2 area (1×10-4 mL). To maintain accuracy, the total cell number counted for each chamber should be between 200 to 500 cells (or 20 to 50 per 1 mm2 square). If the number of cells counted is above this range, dilute your sample to get to this range. (3). Before adding cells to the hemocytometer, make sure that the cells suspension in the media is as homogenously dispersed as possible. (4). Draw out 10 ?L of cell suspension and mixed with equal amount of trypan blue. Trypan blue will stain dead cells blue. (5). Mix well, and then with the hemocytometer and cover glass ready, pipette 10 ?L of the suspension through the groove/opening of the chamber, which would take in the solution through capillary action. Do the same for both sides. (6). Count the number of cells in all nine 1 mm2 squares in each chamber and average the number of cells in each square. When counting, scan across each row of squares within the 1 mm2 area. (7). Apply the formula above to find concentration per mL. Other Articles by Sasha Bakhru:Sasha Bakhru: Brief Introduction to Adult Stem CellsSasha Bakhru: Cellular Microencapsulation History Sasha Bakhru's Bibliography |