A498 Human Renal Cancer Cell Specific Culture Medium Instruction Manual

Product Introduction:

The A-498 cell specific culture medium has been carefully optimized by the technical team, and after long-term testing, this product can maintain the optimal growth state of A-498 cells. This product already contains various ingredients required for the growth of A-498 cells, and no additional ingredients are needed. It can be directly used for in vitro culture of A-498 cells. This product is for further scientific research use only and should not be used for diagnosis, treatment, clinical, household, or other purposes.

Specific steps of cell culture:

1、 Common equipment

1. Equipment in the preparation room

Single distilled water distiller, double distilled water distiller, acid tank, oven, pressure cooker, storage cabinet (for storing unsterilized items), storage cabinet (for storing disinfected items), packaging table. Equipment for the liquid preparation chamber: torsion balance and electronic balance (for weighing drugs)PHMeasurement of culture mediumPHValue), magnetic stirrer (equipped with a solution chamber to stir the solution).

2. Equipment in the training room

Liquid nitrogen tank, storage cabinet (for storing miscellaneous items), fluorescent and ultraviolet lamps, air purifier system, low-temperature refrigerator（-80℃）Air conditioner, carbon dioxide cylinder, side table (write experimental records).

3. Equipment that must be placed in a sterile room

Centrifuge (for collecting cells), ultra clean workbench, inverted microscopeCO2Incubator (incubation culture), water bath, ozone disinfection and sterilization machine4℃Refrigerator (for storage)serumAnd culture medium).

2、 Aseptic operation

（1） Sterilization in a sterile room

1.Regularly clean the sterile room: clean once a week, first use tap water to mop the floor, wipe tables, super clean workbenches, etc., and then use3‰Lai Su Er or Xin Jie Er Mie or0.5%Wipe with peracetic acid.

2.CO2Sterilization of incubator: First use3‰Wipe with Xin Jie Er Mie, then use75%Alcohol wiping or0.5%Peracetic acid, then irradiate with ultraviolet light.

3.Pre experiment sterilization: Turn on the UV lamp, oxygen sterilizer, and air purifier system20-30minute.

4.Sterilization after experiment: use75%Alcohol（3‰Wipe the ultra clean stage, edge stage, and inverted microscope stage with Xinjie'er Mie.

Cell culture method:

01 Primary culture operation steps

The operation steps of primary culture are: sampling→separation→Cultivate.

（1）Sampling: Different tissues have different sampling methods, but the materials should be kept fresh and strictly sterile.

The steps for cell passage culture are as follows:

（1）Observe the cell morphology and growth density under an inverted microscope before passage, and when the cell growth density reaches80%~90%At that time, it can be passaged.

（2）Suck or discard the culture medium from the culture bottle. joinPBSwash1~2Next, gently shake left and right before discarding.

（3）Add an appropriate amount of the culture bottle according to its sizeEDTAGenerally, it should cover the entire bottom of the culture bottle.

（4）Put the culture bottle in37℃ CO2Digest in the incubator,2~5minThen take it out and observe it under an inverted microscope. When it is found that70%~80%When the cells contract and become round, and the intercellular space becomes larger, gently tap the culture bottle to make the remaining cells fall off, and then immediately add2Stop digestion with double the amount of culture medium, gently blow with a pipette, mix evenly to prevent excessive digestion.

（5）Suck out all cell suspensions into centrifuge tubes,1000rpm/mincentrifugation3~5min.

（6）Discard the supernatant, add an appropriate amount of culture medium to resuspend the cells, gently blow and mix well to evenly disperse the cells.

（7）Use a pipette to aspirate an appropriate amount of cell suspension, inoculate it into a new culture bottle at a suitable density, replenish the culture medium, shake well, and place it in37℃ CO2Cultivate in the incubator.

（8）Determine the time for fluid replacement or passage based on the growth status of cells, and even perform cell cryopreservation.

Notes:

（1）Strictly conduct aseptic operations, all reagents and consumables used should be sterile, and must be irradiated with ultraviolet light in a sterile ultra clean workbench30minAbove, to prevent contamination of cells.

（2）The culture medium used must be suitable for cell survival and growth. Cells derived from different animal species and tissue types have varying requirements for the culture medium. If necessary, pre experimental methods can be used to select the appropriate culture medium.

（3）Fetal bovine serum plays a crucial role in maintaining cell survival and promoting cell growth. Suitable fetal bovine serum can be selected based on literature or pre experiments. Once confirmed, it should be kept in use until the experiment is completed.

（4）When digesting cells, it is important to avoid situations where the digestion time is too short, resulting in incomplete digestion, a small number of collected cells, or a prolonged digestion time that causes cells to form clumps and become loose. At this point, the cells may have already been damaged or died, affecting the number of cells and the progress of the experiment.

（5）When centrifuging cells, it is important to avoid insufficient collection of cells due to low centrifugal force, or damage to cells caused by excessive centrifugal force. After discarding the supernatant from the centrifuged cell pellet, the pellet should be first dispersed and then resuspended in culture medium. This method causes less damage to the cells than direct blowing and can improve cell viability.

（6）When blowing cells, it is advisable to avoid the production of cells as much as possible to prevent damage to the cells and affect their state (residual liquid in the suction head during the blowing process can reduce the generation of bubbles).

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