E14 cell specific culture medium

Aseptic operation:Cell culture should be carried out in a sterile environment. Before use, the ultra clean workbench should be sterilized by ultraviolet radiation and the incubator should be cleaned regularly. Experimental personnel need to change their work clothes, shoe covers, wear masks, and gloves.

Equipment sterilization:Glassware, metal instruments, etc. can be sterilized with high-pressure steam, while plastic products are often sterilized with ethylene oxide or sterilized products can be purchased.

Reagent selection:The culture medium, serum, buffer and other reagents used should be sterile and free of contamination, and sterile testing should be conducted before use. Different cells have different requirements for the composition of the culture medium, and the appropriate culture medium should be selected according to the cell type.

Cultivation conditions:The suitable culture temperature for most mammalian cells is 37 ℃, and the deviation should be controlled within ± 0.5 ℃. The incubator should maintain good sealing, and the gas concentration and flow rate should be checked regularly. Cell culture usually requires 5% CO2 to maintain stable pH value of the culture medium. The humidity inside the incubator should be maintained at around 95%.

‌Daily observation:Observe the cell morphology, growth status, and color changes of the culture medium under a microscope every day.

Regular testing:Regularly conduct contamination testing on cells for mycoplasma, bacteria, fungi, and other contaminants.

E14Cell specific culture medium

Product Introduction

E14Cell culture medium is composed ofThe team has carefully optimized and after long-term testing, this product can maintainExcellent growth status of E14 cells.

This product already includes The various components required for E14 cell growth can be directly used for E14 cell culture without adding any ingredients.

Main components of the product

DMEM basic culture medium 405mL

Defined FBS 75 mL

Transportation and storage

Transportation: Low temperature transportation in an insulated box containing biological ice packs

Storage method: 2 ℃~8 ℃, away from light, stored for 2 months; ׏

Please note that it is only for scientific research purposes.

Some components in the culture system are harmful to human health. Please do not use exposed skin to come into contact with the liquid in the culture system or containers containing residual liquid in the culture system; The concentration and harmfulness of these harmful substances are relatively low. If there is contact, rinse immediately with tap water.

revival

1) Preheat the water in the constant temperature water bath to 37 ℃;

2) Prepare a 15ml centrifuge tube, add 5ml of culture medium containing 10% FBS, and preheat it in a 37 ℃ water bath;

3) After putting on goggles and thick woolen gloves, take out the cells to be revived from the liquid nitrogen tank and transfer them to a 37 ℃ constant temperature water bath as soon as possible

Shake the freezing tube for thawing to increase the thawing rate;

4) Transfer the melted cells from the cryovial into a pre prepared centrifuge tube, mix well, and centrifuge at 1000rpm for 5 minutes;

5) Prepare a T-25 culture bottle, write the cell name and date, and add 4ml of culture medium;

6) After centrifugation, discard the supernatant and resuspend the cells in 1ml of culture medium. Transfer to T-25 cell culture, mix well, and then transfer to

Cultivate and let stand in a CO2 incubator.

Passage (recommended for cell passage from one to two)

1) When the cell confluence reaches 85% or above, passage can be performed.

2) In the biosafety cabinet, open the mouth of the culture bottle and collect the culture medium inside the bottle;

3) Add 3ml sterile 1 × PBS to the culture bottle, place the culture bottle horizontally so that PBS can infiltrate all the areas on the bottom of the culture bottle, and discard the PBS;

4) Add 1ml of digestive solution to the bottle, soak the bottom and incubate in a 37 ℃ CO2 incubator for 1-2 minutes;

5) After incubation, observe whether the cells become round and float under an inverted microscope. If all are digested, add 2ml of culture medium containing 10% FBS directly into the culture bottle, and aspirate the suspension into a 15ml centrifuge tube;

① Prepare a sterile 15ml centrifuge tube and add 2ml of culture medium containing 10% FBS;

② Inhale the digested cells into the centrifuge tube in ① for neutralization (avoid blowing ③); Add 1ml to the previously digested culture bottle and continue digestion for about 2 minutes. Gently tap the culture bottle, and about 95% of the cells will be removed.

6) Centrifuge at 1000rpm for 5 minutes;

7) Prepare two new T-25 culture bottles, each containing 4ml of culture medium.

8) After centrifugation, discard the supernatant and resuspend the centrifuged cells in 2ml of culture medium. Transfer the resuspended cells into two T-25 culture bottles, each containing 1ml;

9) Place the culture bottle horizontally, shake and mix well, then place the bottle in a 37 ℃, 5% CO2 incubator for static cultivation.

Cryopreservation (it is recommended to freeze one T25 cell per bottle)

1) 6) Refer to the passage steps

7) After centrifugation, discard the supernatant and resuspend the cell pellet in 1mL of frozen solution, then transfer it to a 1.8ml cryovial;

8) Transfer the cryovial into a program cooling box filled with isopropanol, and then transfer it to a -80 ℃ freezer for overnight cooling;

9) The next day, remove the cryovial from the cooling box and transfer it to a liquid nitrogen tank for storage as soon as possible.

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