293 HEK-293 (human embryonic kidney cells)

293 HEK-293 (human embryonic kidney cells)

aliasHek293; HEK-293; HEK 293; HEK:293; 293; 293 HEK; Human Embryonic Kidney 293

Training Plan A (default)

Growth medium:

MEM (ATCC Improved)+10% FBS+1% P/S

Cultivation conditions:

Gas phase: Air, 95%; CO2， 5%, temperature: 37 ℃

PrecautionsThe cell is loosely attached to the wall, please operate as gently as possible; Preheat the culture medium when changing the solution; If there are large detached cell clusters during receipt, it is a normal phenomenon. Please handle according to the receiving precautions.

Reference materials (source literature)

The background description is the formation of immortalized cells from human embryonic kidney cells transfected with human adenovirus 5 (Ad5), which contains and expresses the transfected Ad5 gene. Early reports indicated that the genome contains DNA from the left and right ends of the adenovirus 5 (Ad5) genome, but now it is clear that only the DNA from its left end exists. After cloning and sequencing of the insertion site of Ad5, it was found that linear nucleotides 1-4344 of Ad5 were integrated into chromosome 19 (19q13.2). The host of human adenovirus vector amplification can express abnormal cell surface receptors of fibronectin, consisting of integrin β 1 subunit and fibronectin receptor α - v subunit.

Age (gender)fetus

Source of organizationKidney; Transform with adenovirus 5DNA

cell typetransformed cell line

Biosafety levelBSL-2

Doubling time~24-30 hours

tumorigenicityYes, forms tumors in nude mice.

Receptor expression statusvitronectin

Preservation institutionATCC; CRL-1573 ATCC： PTA-4488 DSMZ： ACC-305 ECACC： 85120602

Cell processing:

1) Resuscitation of frozen cells: Quickly shake and thaw the frozen tube containing 1mL of cell suspension in a 37 ℃ water bath, then add it to a centrifuge tube containing 4-6mL of culture medium and mix evenly. Centrifuge at 1000RPM for 3-5 minutes, discard the supernatant, and resuspend the cells in the culture medium. Then add the cell suspension to a culture bottle (or dish) containing 6-8ml of culture medium and culture overnight at 37 ℃. The next day, observe cell growth and cell density under a microscope.

2) Cell passage: If the cell density reaches 70% -90%, passage culture can be carried out. This cell is a suspended and slightly adherent cell, and passaging can refer to the following methods:

1. Collection: Collect the suspended cells in the culture bottle into a centrifuge tube. Wash cells 1-2 times with PBS that does not contain calcium or magnesium ions. Due to the weak adhesion of cells to the wall, cells will fall off after PBS washing, so PBS also needs to be recovered into centrifuge tubes.

2. Add 0.25% (w/v) 0.53 mM EDTA to culture bottles (T25 bottle 1-2mL, T75 bottle 2-3mL) and digest in a 37 ℃ incubator for 1-2 minutes (for difficult to digest cells, the digestion time can be appropriately extended). Then observe the digestion of the cells 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 3-4ml of culture medium containing 10% FBS to terminate digestion.

3. Centrifuge the collected suspended cells, cells in PBS cleaning solution, and digested adherent cells at 1000rpm for 5 minutes, discard the supernatant, add 1-2mL of culture medium, resuspend and mix well, and then divide the cell suspension into new T25 bottles at a ratio of 1:2. 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.

3) Cell cryopreservation: It is recommended to freeze a batch of cell seeds during the first 3 generations of culture after receiving the cells for subsequent experimental use. Taking T25 bottles as an example:

1. When freezing cells, collect digested cells into centrifuge tubes according to the process of cell passage, and use a hemocytometer to determine the freezing density of cells. The recommended freezing density for general cells is 1 × 10 ⁶~1 × 10 ⁷ live cells/ml

2. Centrifuge at 1000rpm for 3-5 minutes and remove the supernatant. Resuspend the cells in the prepared cell cryopreservation solution, and distribute them into a cryopreservation tube at a concentration of 1 × 10 ⁶~1 × 10 ⁷ live cells/ml per 1ml of cryopreservation solution. Label the cells with their names, generations, dates, and other information.

3. Place the cells to be frozen in a programmed cooling box, refrigerate at -80 degrees Celsius overnight, and then transfer them to a liquid nitrogen container for storage. Simultaneously record the position of the cryovial in the liquid nitrogen container for future reference and use.

Operation steps:

Step 1: Take out the serum-free non programmed cryopreservation solution from the refrigerator and set it aside for later use

Step 2: Collect logarithmic growth stage cells by centrifugation (digest adherent cells and centrifuge, suspend cells and centrifuge directly at 1200rpm or 250g for 3 minutes)

Step 3: Discard the supernatant, add an appropriate amount of serum-free non programmed freezing solution, and resuspend the cells

Step 4: Divide the tubes into cryovials at a rate of 1-1.5mL/tube, tighten the tube caps, and mark them properly

Step 5: Transfer the cryovial directly into a -80 ℃ freezer and after 24 hours, transfer it to liquid nitrogen for long-term storage

When there is no freezing box or non programmed freezing solution, manual gradient cooling can be selected. However, manual gradient cooling is not suitable for all cells and the effect is unstable, so it is also necessary to conduct cryopreservation testing first.

Notes:

1. After receiving the cells, if you find that the dry ice has evaporated completely, the cryovial cap has fallen off, is damaged, or the cells are contaminated, please contact us immediately.

2. After receiving the cells, do not open the bottle cap. Wipe the bottle with alcohol and place it in the incubator for 2-4 hours (depending on the cell density) to stabilize the cell state. Next, observe the cell growth under an inverted microscope and take photos of the cells at different magnifications (it is recommended to take a photo of the overall appearance when collecting the cells, observe the color of the culture medium and whether there is any leakage, and then take a photo of the cell status under the microscope, one at 100 * and one at 200 *). Observe and record whether there is any contamination during the transportation of the cells. As a basis for our sales.

3. Due to various factors such as environment, operation, and transportation affecting cell status, our company only guarantees the cell status of customers within one week after receiving the cells. Therefore, when customers need after-sales service, they need to provide proof of the time of receiving the cells and proof of the time of receiving and communicating with customer service personnel after discovering the problem. The interval between periods cannot exceed 7 days.

4. All animal cells are considered to have potential biological hazards and must be operated in a secondary biosafety platform. Please pay attention to protection, and all waste liquids and containers that have come into contact with these cells must be sterilized before disposal.
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