Cell Spreading Technology
Cell Spreading Technology
Summary
Cell spreading technique is a common and necessary experimental operation for cells, essential in cell counting, culture, immunofluorescence and other experimental operations. According to the experimental purpose of a certain concentration of cell dilution will be inoculated into the cell counting plate without contamination, convenient for the next operation.
Operation method
Cell spreading (detailed steps)
Materials and Instruments
75% alcohol, alcohol swabs, ultra-clean table, inverted microscope, alcohol lamp, pipette gun, tip, PBS solution, 0.25% trypsin, Petri dish, fresh medium with 5% serum, 15 mL centrifuge tube, centrifuge. Move 1、Preparation: Before the experiment, open the ultra-clean table 30 minutes in advance, open the water bath, preheat the medium, PBS and trypsin at 37 ℃. After UV irradiation for 30 min: After 30 min of UV irradiation, wipe your hands with 75% alcohol and wipe the ultra-clean table with alcohol cotton balls; ② Sterilize the culture medium and materials needed for the experiment (except serum and culture medium) by putting them into the ultra-cleaning table; ③ Inverted microscope, observe the state of the cells, whether they have grown full of culture bottles, need to be divided. 2. Discard the culture medium and try to remove the medium with a gun. Add 5 mL of PBS liquid along the cell-free wall to clean it once, and shake it in four directions to pour it out. 3、Add 0.25% pancreatic enzyme 600 uL into the petri dish, spread it evenly at the top and bottom, and digest it at 37 ℃. Digest the cell line for about 1 min, and digest the primary cell for about 5 min, and observe it at any time, and when you see the cell slurry left, it is digested completely. 4, add 4 mL of fresh medium containing 5% serum, blow the digested cells repeatedly to make them off the wall and dispersed, and make cell suspension, and then load it into a 15 mL centrifuge tube. 1000 rpm centrifugation for 3 min. 5. Discard the supernatant. Wash the cells with 3 mL of PBS, blow and mix, wash twice, centrifuge at 1000 rpm for 3 min, and discard the supernatant. 6. Add 3 mL of fresh medium with 5% serum to the cell sediment and mix well by blowing to obtain the diluted cell suspension. 7、Pipette 15 µL of cell suspension, slowly fill the cell suspension along the edge of the coverslip between the coverslip and the counting plate, taking care that there are no air bubbles in the coverslip. Calculate the total number of cells counted in the four compartments. For the pressure line cells, the technical principle is to count the top but not the bottom, and to count the left but not the right. Calculation formula: number of cells / mL = total number of cells in the four compartments / 4 × 104. 8, according to the density of the plate, calculate the amount of diluted cell suspension, the remaining volume of cell dilution solution to make up. Caveat 1、Cell plates do not need to be moistened with culture medium in advance, directly drop in the medium mixed with cells from the side;2、After placing it for 30 s~1 min, shake it back and forth for 5~8 times;3. Place the plate on the horizontal table and let it stand for 2~5 min, then put it into the incubator.4、Because some cell scraper is not good to use, there is no way to scrape the surrounding, generally in the extraction of protein or RNA sometimes want to lay more in the middle, less around the plate, the technique is as follows: cell plate does not need to be rinsed in advance, from the middle of the drop into the plate; and then, rotate the plate clockwise or counterclockwise in one direction for 3~5 turns; and then in the horizontal table to rest for 2~5 min; and finally put into the incubator. incubator.5, cell counting before and after a large error to consider the cell settlement led to uneven suspension; suspension volume is too large or too small; dilution is too high or too low and other reasons.6、Try to blow the cells as single as possible to prevent clumping, and use the pipette gun to blow sufficiently, so that they are evenly suspended in the medium.7、Due to the amount added to the counting chamber, too little will lead to bubbles in the counting chamber, too much will make the coverslip on the counting plate not close to the counting plate, making the height higher and the volume larger; the counting chamber volume is 9 mm3The volume of the counting chamber is 9 mm 3 , so generally add about 15 uL.8、When counting, the optimal concentration of cell dilution is 5~10×105 cells / mlL.The optimal concentration of cell dilution is 5~10×10 5 cells/mlL, such as general 10 cm dish has about 3~5 million cells, some cells can reach 10~20 million even if they are small, according to the required number of cells, some cells can be taken out for dilution or counted after continuous dilution. For more product details, please visit Aladdin Scientific website.