Operating procedure
First, recovery
1. The treatment of the culture dish/plate is carried out in advance using Matrix. The Matrix is ​​usually stored at -20 ° C and thawed in a 4 ° C refrigerator or ice before use. After the Matrix was thawed, it was quickly diluted with PBS liquid at a ratio of 1:100. Add 1 ml per well of a 6-well plate, add 2 ml of 150 px dish, add 3 ml of 250 px dish, and shake the dish/plate quickly after the addition, so that the added Matrix completely covers the entire bottom of the dish. Place in a 37 ° C incubator for 4 hours, remove the coating solution before use (if not used temporarily, seal with a sealing film, can be stored in the refrigerator at 4 ° C for one week).
2. Prepare iCell Thawing Complete Medium (iCell Thawing Base Medium and iCell Thawing Additive) before resuscitation. Add an appropriate amount of thawed liquid (2 ml per well, 6 ml plate, 3 ml, 150 px dish and 9 ml), add 10 ug/ml of Y27632 factor, and place in a 37 ° C incubator.
3. Resuscitation of a cell with 5 ml of the above mixed medium, fully preheated in a 37 ° C water bath before resuscitation. After removing the cryotube from the liquid nitrogen tank, transfer it to the biosafety cabinet quickly (if the position of the liquid nitrogen tank is far from the safety cabinet, transfer the cryotube to the safety cabinet with a container filled with liquid nitrogen) and heat it. A good complete medium is thawed (into the cryotube with a pipette) - the medium is withdrawn and the dissolved cells are exchanged until completely dissolved.
4. Centrifuge at 200Xg for 5 minutes, remove the supernatant, add 1 ml of iCell complete thawing medium (containing 10 ug/ml of Y27632 factor), and use a finger to break the tube. The iCell was completely thawed into the centrifuge tube by inoculating 1 ml per plate/dish, and gently pipetted three or four times before adding to the dish/plate. The horizontal cross vibration allowed the cells to be evenly distributed, and cultured in a 5% CO 2 , 37 ° C incubator for 24 hours.
5. After 24 hours, switch to iCell complete medium (iCell basal medium and iCell basal medium additive). Change the fluid every 22-24 hours later. Cells aged 80-90% need to be passaged or frozen.
Second, passaging / frozen
1. Before the passage, the treatment of the culture dish/plate should be carried out in the same way as in the case of recovery. The medium was iCell complete medium, and 10 ul/ml of Y27632 factor was added.
2. Before passage/freezing, prepare a calcium-free magnesium ion PBS solution and a passage working solution (0.5 mM EDTA + 0.025% trypsin) preheated at 37 °C.
3. Aspirate the iCell complete medium and wash it twice with a pre-warmed PBS solution at 37 °C.
4, add appropriate amount (according to 6-well plate per well plus 1ml, 150px dish plus 2ml, 250px dish plus 3ml amount) of 37 ° C preheated good passage of working fluid.
5, place at 37 ° C for 4-5 minutes (take it out after the first minute, use the finger to flick the bottom of the plate / dish), under the microscope, observe that most of the cloned edges begin to separate from the culture dish / plate bottom, and clone most of the inside. There was a gap between the cells but not separated from each other. It was observed by the naked eye that the cell colonies became opaque and whitish, indicating that the cell digestion time was ideal.
6. Quickly aspirate the passage solution, add appropriate amount of iCell complete medium to terminate digestion, and use a pipette to blow the bottom of the dish/plate (not more than 10 times), so that the attached cell colonies fall off. (If the digestion time is not proper, causing the cells to completely peel off from the dish/plate bottom, do not aspirate the passage, add the same amount of iCell complete medium to terminate the digestion).
7. Move into the centrifuge tube, resuspend after centrifugation. The passage ratio is 1:8-1:12; frozen storage is 1 per well in a 6-well plate, 2-4 in a 150px dish, and 6-12 in a 250px dish. 0.8 ml of 90% iCell complete medium and 10% DMSO were added to each.
8. When recovering, proceed according to the ratio of 1:4 to 1:6 before freezing.
Precautions
1. Observe the cell growth and cell morphology changes and take photos of the cells each time the liquid is changed. However, do not operate too long outside the incubator to avoid adverse effects on cell growth due to temperature.
2. Before the medium is changed, the new medium should be preheated at 37 °C. In order to avoid the influence of various components in the medium caused by repeated heating of the medium, only the amount to be replaced is preheated.
3. Pipette the cells with a pipette because the end of the pipette is smoother and less harmful to the cells than with a 1 ml gun.
4. When the cell density reaches 80-90%, it should be passed in time, otherwise it will cause changes in cell morphology. However, if the cells are unevenly mixed during plating and a part of the colonies are formed too large, passage is carried out even if it is not 80-90%.
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