Cell Technology Topic: Introduction to Cell Passage Experiment

1. Digestion of adherent cells:

(1) Aspirate or pour off 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) The 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, and the cell gap should be terminated immediately after the cell gap is enlarged.

(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, leaving the cells out of 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 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 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.