African clawed frog kidney cell ACTK-1 (species identification)

1African clawed frog kidney cell ACTK-1 (species identification)culture conditions

IIAfrican clawed frog kidney cell ACTK-1 (species identification)Post delivery processing

Recovery cell processing method:The best way to transport cells is to fill the culture medium and seal the bottle mouth after cultivating to a good state. Received cells for useSpray 75% alcohol throughoutcellbottle，Strictly sterileafterPlace the cell vial in the incubator and let it stand2-4 hours to stabilize the cell state.After standingObserve cell growth under a microscope and take photos of the cells at different magnifications for preservation（best40x, 100x, 200x (one sheet each)，Photos from the previous three daysisImportant after-sales basis, if no photos are provided, it is assumed that the received condition is good.

Cryopreservation cell processing method:After receiving the cells, observe whether there is any residual dry ice in the foam box, whether the cryopreservation tube is intact, and whether there is thawing. If the dry ice is vaporized or the cryopreservation tube is thawed, please take photos in time and contact us. If the cells do not contact us within three days of receiving the goods, it will be assumed that the goods have been received well. If there are any issues with the activity of the first tube during resuscitation, please contact us promptly. Our technical personnel will communicate with you for guidance before resuscitation of the second tube,Unauthorized recovery of the second pipe without contacting us will result in no after-sales service.

Cell culture steps

a、subculturing：ifnot exceedingAt 80% confluence, collect the bottled culture medium into a centrifuge tube, leave 5ml of culture medium, and place it in 37 ℃, 5% CO₂Incubator cultivation;If the cell densitysuper80%, can be passaged for cultivation.

adherent cellsThe steps are as follows:

1) Discard the culture supernatant and rinse the cells 1-2 times with PBS without calcium or magnesium ions;

2) Add 1mL of digestion solution (0.25% Trypsin-0.53mM EDTA) to a culture bottle, digest in the incubator for 1 minute, and then observe the cell digestion under a microscope. If most of the cells become round and fall off, quickly take them back to the operating table, tap the culture bottle a few times, and add 6ml of 10% serumculture mediumTerminate digestion;

3) Gently blow the cells, remove them, and aspirate them into a centrifuge tube. Centrifuge at 1000 rpm for 5-8 minutes, discard the supernatant, add 1-2 mL of culture medium, and blow evenly;

4) Add 5-6mL of culture medium to each bottle, and divide the cell suspension into new 6cm culture dishes or T25 culture bottles containing 5-6 mL of culture medium in a ratio of 1:2.

(i.eInoculate one T25 bottle into two T25 bottles or two 6cm culture dishes

Suspended cellsThe passage steps are as follows:

1. Semi liquid exchange method

Half liquid exchange treatment, place the culture bottle vertically in the incubator and let it stand stillAfter about 1 hour, gently aspirate about 3ml of culture medium, and then replenish 3ml of culture medium. If the color of the culture medium changes slowly, add about 500ul of FBS directly. When passaging, you can directly supplement 5ml of culture medium and divide it into two culture bottles for cultivation. Generally, after passaging for about 3 times, centrifuge once to remove dead cells.

2. Centrifugal liquid exchange method

If it is necessary to divide the bottles, the cell suspension can be collected into centrifuge tubes1000rpm， Centrifuge for 5 minutes, discard the supernatant, add 1-2 mL of culture medium, resuspend and mix well. Then, divide the cell suspension into new T25 bottles at a ratio of 1:2. Add 6-8 mL of new culture medium prepared according to the instructions to maintain the growth vitality of the cells. Subsequent passages should be carried out according to the actual situation at a ratio of 1:2~1:5.

b、Cell cryopreservation:

1. When the cells grow to cover 80% of the surface area of the culture bottle, discard the culture medium in the T25 culture bottle and wash the cells once with PBS;

2. Add about 1ml of 0.25% digestion solution to the culture bottle, observe under an inverted microscope, and wait for the cells to shrink and become round before adding 5ml of culture solution to terminate digestion. Gently blow the cells to detach, then transfer the suspension to a 15ml centrifuge tube and centrifuge at 1000rpm for 5 minutes;

3. Discard the supernatant, precipitate the cells and add 1ml of Yaji Biological Serum Free Cryopreservation Solution（Item No.C7001）， Mix well and add to the cryovial.

4. Put the frozen cells directly into-A refrigerator at 80 ℃ is sufficient for later usewantTransfer the cells into a liquid nitrogen tank,thenNeed to be in-80℃ freezermiddlestoreOver 24 hoursagainTransfer to liquid nitrogen tank.

C、Cell revival:

1Remove the cell cryopreservation tube from liquid nitrogen（attentionwearprotectionMask), quickly place it inside Thaw in a 37 ℃ water bath until there are no crystals in the cryovial, then wipe the outer wall of the cryovial with 75% alcohol;

2Transfer the cells in the cryovial to a container containing Centrifuge at 1000rpm for 5 minutes in a 15ml centrifuge tube containing 5ml of culture medium;

3Discard the supernatant and use for sedimentationResuspend 5ml of culture medium and inoculatetoT25 culture bottle,putYu37℃,5%CO₂Cultivate in a cell culture incubator;

4The next day, switch to fresh culture medium to continue cultivation.

Notes:Some cells are not firmly attached to the wall and are prone to cell detachment during transportation, which is a normal phenomenon. If there is a significant amount of detachment, collect all the culture medium from the culture bottle into a centrifuge tube,Centrifuge at 1000rpm for 5 minutes,collect the supernatantTransitional cultivation(Comparative cultivation in later stage)Add sediment1-2ml， Gently blow, resuspend, digest for 1-2 minutes, then add 5ml of culture medium to terminate the reaction. Centrifuge again, discard the supernatant, and resuspend in 1-2ml of culture medium. Then divide the cells into two T25 cells and subculture them in a 1:2 ratio. Add new culture medium to 5-8ml per cell, and finally place them in 37 ℃, 5% CO solution₂Cultivate in a cell culture incubator.