Human skin fibroblast HFF1 (STR)

1、 Human skin fibroblast HFF1 (STR)culture conditions

IIPost 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. Spray the entire cell with 75% alcohol upon receiptCell bottles,Strictly sterileafterPlace the cell bottle in the incubator and let it stand for 2-4 hours to stabilize the cell stateAfter standing still, observe the growth of cells under a microscope and take photos of the cells at different magnifications for preservation (preferably one 40x, one 100x, and one 200x each). The photos taken in the first three days are important after-sales reference, and if no photos are provided, it is assumed that the cells have been received in good condition.

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、subculturingIf:When the confluence degree does not exceed 80%, 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 density exceeds 80%, subculture can be carried out.

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.e. one T25 bottle is passaged and inoculated into two T25 bottles or two 6cm culture dishes)

suspended cellsThe passage steps are as follows:

1. Semi liquid exchange method

Half change the medium treatment, place the culture bottle vertically in the incubator for about 1 hour, gently suction off about 3ml of culture medium, and then replenish 3ml of culture medium. If the color change of the culture medium is slow, add about 500ul of FBS directly. When passaging, add 5ml of culture medium directly 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 necessary, the cell suspension can be collected in a centrifuge tube at 1000rpm and centrifuged for 5 minutes. Discard the supernatant, add 1-2mL of culture medium, resuspend and mix well. Then, divide the cell suspension into new T25 bottles at a ratio of 1:2 and add 6-8ml 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 (SKU: C7001), mix well, and add to the cryovial.

4. Simply place the frozen cells directly into a -80 ℃ freezer. If the cells need to be transferred to a liquid nitrogen tank later, thenNeed to be refrigerated at -80 ℃middleStore for more than 24 hoursTransfer it back into the liquid nitrogen tank.

C、Cell revival:

1. Remove the cell cryopreservation tube from liquid nitrogen (wear protective gear)Mask), 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, resuspend the precipitate in 5ml of culture medium, and inoculateTo T25 culture bottle, placeAt 37 ℃, 5% CO₂Cultivate in a cell culture incubator;

4. The 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 lot of detachment, collect all the culture medium in the culture bottle into a centrifuge tube and centrifuge at 1000rpm for 5 minutes,collect the supernatantTransitional cultivation(Comparative cultivation in later stage)Add 1-2ml of precipitate, gently blow and 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 add it to the cells37℃,5%CO₂Cultivate in a cell culture incubator.

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