According to the cell growth defects, there are three methods of subculture: suspension growth cell passage, semi-suspended growth cell passage (Hela cells), adherent growth cell passage.
experimental method
Cell culture technique
Digestive method
Experimental Methods Principles Cultured cells are passaged according to different methods of different cells. Adherently grown cells are passaged by digestion; some adherently grown cells can be passaged by direct pipetting; cells that are suspended for growth can be passaged by direct blown or centrifugal separation, or the supernatant can be removed by natural sedimentation, and then blown down. .
Experimental Materials
cell
Reagents, kits
D-Hanks liquid calf serum RPMI1640 double antitrypsin EDTA NHCl NaHCO3
Instruments, consumables
Purification Workbench Centrifuge Constant Temperature Water Bath Refrigerator Inverted Phase Difference Microscope Incubator Straw Glass Bottle Culture Bottle Waste Liquid Cylinder Tip Head Rubber Plug Centrifuge Tube Volume Adding Gun Red Blood Cell Counting Plate
Experimental procedure
1. Digestion of adherent cells:
(1) Aspirate or drain the old culture solution in the bottle.
(2) D-Hanks is washed 2 to 3 times.
(3) Add an appropriate amount of digestive juice (trypsin or mixed with EDTA) to the flask and gently shake the flask to allow the digestive juice to flow through all cell surfaces.
(4) Then suck or pour off the digestive juice, then add a proper amount of new digestive juice, gently shake and then pour off most of the digestive juice, leaving only a little for digestion (you can also use the above steps to directly digest the digestive juice for digestion).
(5) Digestion is preferably carried out at 37 ° C or at room temperature of 25 ° C or higher.
(6) After digesting for 2 to 5 minutes, the culture flask was placed under a microscope to observe the cytoplasm retraction. After the cell gap was enlarged, the digestion should be terminated immediately.
(7) Aspirate or pour off the digestive juice. If it is digested with EDTA, add a few milliliters of Hanks solution and gently rotate the culture flask to wash away the residual digestive juice.
(8) Add the culture solution. If only trypsin can be used directly, add a little serum-containing medium to stop digestion.
(9) Using an elbow pipette, sucking the culture medium in the bottle, repeatedly blowing the cells on the bottle wall, and the blowing process should be carried out sequentially.
(10) From the bottom of the bottom of the flask to the end of the other side, to ensure that all the bottoms are blown, so that the cells are separated from the bottle wall to form a cell suspension. When blowing, be gentle and don't use too much force.
(11) Count and inoculate separately in new culture flasks.
2. Passage of suspended cells: Suspension cells can be collected by centrifugation, passaged, or directly passaged.
Centrifugal passaging:
(1) Transfer the cells together with the culture solution to a 15 ml centrifuge tube.
(2) Centrifuge at 800~1000 rpm for 5 min.
(3) Discard the supernatant, add new culture solution to the centrifuge tube, and blow with a pipette to form a cell suspension.
(4) Count and inoculate separately in new culture flasks.
(5) Direct passage means that the suspended cells are slowly precipitated at the bottom of the bottle, and the supernatant is aspirated by 1/2 to 2/3, and then the cell suspension is formed by pipetting to form a cell suspension.
3. Passage of partially adherent cells
(1) Some cells that are not well-attached, such as Hela cells, can be detached from the wall without being digested and directly passed down.
(2) For most of the adherent cells, the cells are damaged by direct blows, and the cells often have a large number of losses, so they need to be digested before they can be beaten.
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Precautions
1. Trypsin should be pre-warmed at a temperature of about 37 °C.
2. The centrifugal speed should be suitable, the rotation speed is too low, the cells can not be effectively separated, the centrifugal speed is too large, and the time is too long, the cells will be squeezed to cause damage or even death.
3. It is necessary to observe the cells regularly and find that the pollution should be treated in time.
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