Human endometrial cancer cell Isshikawa culture instructions (updated in 2022)

Human endometrial cancer cells Isshikawa culture instructions

First, the cell culture conditions

Second, the cells received after treatment
After the culture is in good condition, filling the complete culture solution and sealing the bottle mouth is the best way
to transport the cells.
The received cells are sprayed with 75% alcohol throughout the cell bottle, sterilized and placed in an ultra-clean table for strict sterility, and the cells are placed in a 37 °C, 5%_CO2 incubator for 3-4 hours to stabilize the cell state before processing
.
Microscope to observe the growth of cells, and the cells in different multiples of the photo preservation (preferably 40x, 100x, 200x each), the first three days of photos are an important after-sales basis, do not provide photos by default received in good
condition.
(Note that the lid should be loosened when the sealed culture flask is placed in the incubator, and after passage, it is recommended that one bottle use the complete medium of the original bottle, and the other bottle use the complete medium of its own supply for comparative culture)Third, the cell culture step
a, cell passage: If the total culture solution in the bottle is not exceeded 80%, collect the complete culture solution in the bottle into the centrifuge tube, leave 5 ml of complete medium, and put in 37 °C, 5%_CO2 Incubator culture; If the cell density exceeds 80%, the subculture can be performed, and the specific steps of the subculture are as follows:
Discard the culture supernatant and wash the cells 1-2 times
with PBS without calcium and magnesium ions.

Add about 1-2 ml of 0.
25% trypsin digestion solution to the culture flask, place in a 37 °C incubator for digestion for 1-2 min, and then observe the cell digestion under the microscope, if most of the cells become round and fall off, quickly take back to the console, tap the culture flask a few times and add more than 5 ml of complete medium to stop digestion
.

3.
Gently pipette the cells so that they are completely shed off before aspirating, then transfer the suspension to a 15 ml centrifuge tube, centrifuge at 1000 RPM for 5 min, discard the supernatant, and add 1-2 mL of complete medium to resuspend
.

The cell suspension is passed in vials (two T25 vials) in a 1:2 ratio, supplemented with new complete medium to 5-8 ml/vial, and finally placed in a cell culture incubator at 37 °C, 5%_CO2
.

b.
Cell cryopreservation:
1.
When the cells grow to 80% of the area covering the culture flask, discard the culture medium in the T25 culture flask and wash the cells with PBS once;
2.
Add about 1 ml of 0.
25% trypsin digestion solution to the culture flask, invert the microscope for observation, add 5 ml of complete culture medium to stop digestion after the cells retract and become round, and then gently pipette the cells to make them fall off, and then transfer the suspension to a 15 ml centrifuge tube, centrifuge at 1000 rpm for 5 min;
3.
Discard the supernatant, add 1ml of Yaji Biological Serum-free Cryopreservation Solution (cargo number: C7001), mix well and add it to the
cryopreservation tube.

4, the cryopreservation cells directly into the -80 °C refrigerator can be, if the cells to be transferred to the liquid nitrogen tank in the later stage, you need to store in the -80 °C refrigerator for more than 24 hours and then transfer to the
liquid nitrogen tank.

C.
Cell recovery:
1.
Remove the cell cryopreservation tube from the liquid nitrogen (pay attention to wearing a protective mask), quickly place it in a 37 °C water bath to thaw it until there is no crystallization in the cryopreservation tube, and then wipe the outer wall of the cryopreservation tube with 75% alcohol;
2.
Move the cells in the cryopreserved tube to a 15 ml centrifuge tube containing 5 ml of complete medium and centrifuge at 1000 rpm for 5 min;
3.
Discard the supernatant, resuspend the pellet with 5ml complete medium, inoculate to T25 culture flask, and place in a 37 °C, 5%_CO2 cell culture incubator;

 
Fourth, precautions:
Individual cells are not firmly attached to the wall, and cell shedding occurs during transportation, which is a normal phenomenon
.
Please collect all the culture medium in the culture flask into a centrifuge tube, centrifuge at 1000 rpm for 5 min, collect the supernatant for transition culture (post-contrast culture), add 1-2 ml of pancreatin to the pellet, gently pipett, resuspend, digest for 1-2 min, add 5 ml of complete medium to terminate the reaction
.
Re-centrifuge, discard the supernatant, add 1-2 ml of complete medium to resuspend
.
Then perform vials (two T25s) in a 1:2 ratio, supplement with new complete medium to 5-8 ml/bottle, and finally place in a 37 °C, 5%_CO2 cell culture incubator
.
V.
After-sales clauses:
1) What are the situations in which cells have problems and can be recurded? What are the criteria for judging?
1.
Various problems encountered during cell transportation, cell loss, bottle damage, serious leakage of culture solution, etc.
, recurrence;
2.
Cell contamination problem, please give us the real experimental results within 48 hours of receiving the product, verify and resend;
3.
After 24 hours of standing cells shipped at room temperature, and 24 hours after the recovery of dry frozen cells shipped, the vast majority of cells did not survive, (real and clear cell status photos should be provided), and reissued;
4.
Dry frozen cells shipped after 24 hours after recovery or at room temperature shipment of cells are left standing for 4 hours and unopened, pollution occurs, recurrence;
5.
For cell activity problems, please give us the real experimental results within 7 days of receiving the product, identify the cell viability with trypan blue staining, and resend after verification;
6.
Please take pictures on the day of receipt of the cells and on the 2nd and 3rd days, and if you are not informed within 3 days, it is deemed that the product is qualified
.
If there is a problem within 4-7 days, there is a detailed step of providing the photo of the cell 3 days before receiving the cell and the photo of the cell problem and the cell-related operation, and communicating with the technician, which is determined by the technician to be our responsibility, reissue
.
If the technical personnel determine that the two parties are liable, the two parties shall negotiate or reissue
it at a rate of 50% of the contract price.

2) What are the cases in which the cells have problems and are not repeated?
1.
The customer causes cell pollution and does not recur;
2.
The customer's incorrect operation causes the cell state to be bad and not to recur;
3.
The cell state caused by the cell culture system recommended by this library is not good and does not recur;
4.
If the cell state is not good, and no photo of the first 3 days of cell culture is provided, it will not be reissued;
5.
Cells that have undergone other treatments during culture will not be recur;
6.
Within 2 days of receiving the cells, they are not informed and do not recur;
7.
Subject to availability
.