In order to do experiments, cell spreading is a common one. However, many people are not very successful, and the distribution is uneven: either dense in the middle and sparse around, or dense around and bald in the middle. The following is the use of a 96-well plate to spread the cells evenly, I hope it will be useful to everyone!
Method/Step
1. Generally, 100 microliters of cell suspension is added to each well of a 96-well plate. Add the cell suspension from the left side of the well near the bottom. After adding half of the plate, mix the cell suspension, do not add it, continue adding the remaining half of the plate. After adding all the boards, cover the board, gently hold the left side of the board with your left hand, and gently tap the right edge of the board with your right hand, paying attention to the grip (I usually tap three times). If it is too strong or too many times, the cells will be concentrated and accumulated. Rotate the plate clockwise (counterclockwise is not good), tap the remaining three sides one by one, let it sit for about 5 minutes, and put it in a 37-degree incubator.
For 6-well plates, 12-well plates or 24-well plates, I add a small amount of serum-free medium to the first well, shake and penetrate to the bottom of the entire well, and then use a pipette gun to suck the bottom of the entire well to the first well. In two holes. Use the same method to penetrate to the bottom of the well while simulating other wells, so that the entire bottom of the well is wet, and the cell suspension will lie flat on the bottom of the well. When adding the cell suspension, it can avoid adding more cells in the middle and adding fewer cells in the middle, and the cells are dispersed more evenly. Note that after adding the cell suspension, the cell suspension should be placed on the workbench to stand still. This method is a bit slow, but it is not slow when you are proficient. It can also be applied, but the strength is not as good as the 96-well plate, and the effect is not as good as the 96-well plate, so I gave up the method of penetrating the bottom of the well.
2. After adding the cell suspension, lift the cell culture plate and observe the light from the bottom up to see if the cells have aggregated. Then tap from the bottom to disperse it.
3. If there is a flat-plate oscillator in the laboratory, it is recommended to use this instrument to oscillate slightly. The effect is good, that is, the amplitude is small and the frequency is high.
4. The cells should be dispersed as much as possible, and most of the cells are in a single state. After centrifugation, it is completely suspended! Some transfer to the six-well plate is shaking! When shaking, don't let the cell fluid turn, otherwise the cells will all be brought to the center and uneven!
5. When a bottle of cells is full, proceed to normal processing. Blow it evenly into the culture flask, and then spread a 6-well plate with 2ml per well. After smearing, wipe it with alcohol cotton without observation, and then put it in the incubator. Slightly three laps to the left, three laps to the right, first three laps, then three laps. Basically, after 24 hours, the cells in each well will be very uniform.
6. Calculate all the required liquid and the number of cells, mix them evenly, and add them to the six-well plate. The six-hole plate fluctuates horizontally in a figure of eight. Observe under the microscope. If it is not uniform, shake again as described above. If the cells are not counted and planted directly, shake the mixing bottle at any time during the process of planting the six-well plate. I usually turn the bottle clockwise or counterclockwise.
7. Move up and down first, then move left and right on the horizontal plate, 5-6 times in each direction, but the key is to put it directly into the incubator after shaking, don't do too much exercise, such as looking in the mirror, otherwise it will be easy to be in the middle Get together again.