Transportation and storage

Dry ice transportation and recovery of viable cells:

1.1mL cryovials are packaged with dry ice for transportation. After receiving them, they are stored overnight in a -80 degree freezer and then transferred to liquid nitrogen or directly resuscitated. 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. T25 bottles of revived surviving cells will be shipped at room temperature and processed according to the procedures for cell reception upon receipt.

Processing after cell reception

1.After receiving the cells, disinfect the bottle wall with 75% alcohol and place the T25 bottle in a 37 ℃ incubator for about 2-3 hours. If you find that the culture bottle is damaged, there is liquid overflow, or the cells are contaminated, please take photos and contact us promptly.

2. Please confirm the cell status under a 4 or 5X microscope, and take 2-3 photos (10 ×, 20 ×) of the newly received cells, as well as one photo of the appearance of the culture bottle, for retention as a basis for the cell status upon receipt during after-sales service.

3. Semi adherent cells and loosely adherent (suspended) cells: Place the T25 bottle in a 37 ℃ incubator for about 2-3 hours and observe the condition of the cells under a microscope. If the cell density is below 60%, the customer needs to collect the suspended cells in the T25 bottle, centrifuge them, resuspend them in complete culture medium, and then transfer them back to the original culture bottle for further cultivation. If the cells grow 70% -90%, the cells need to be passaged. During passaging, the suspended cells in the culture medium need to be collected, centrifuged, and recovered.

4. Note: The transport medium (infusion medium) cannot be used to culture cells anymore. Please use a newly prepared complete medium according to the instructions for cell culture conditions to culture cells. After receiving the cells, it is recommended to subculture them for the first time in a T25 culture bottle at a ratio of 1:2.

Preparation of culture medium and culture cryopreservation conditions

1.DMEM basic medium 87%, high-quality fetal bovine serum 10%, GlutaMAX-1 glutamine 1%, HEPES 1M buffer solution 1%, P/S penicillin streptomycin 1%.

2. Cultivation conditions: Gas phase: air, 95%; Carbon dioxide, 5%. Temperature: 37 degrees Celsius, humidity in the incubator is 70% -80%.

3. Suggested cryoprotectant formula: 90% serum, 10% DMSO, ready to use and prepare. Attention: All commercial cryoprotectants may not be suitable for this cell, please use with caution.

Cell revival, passage, and cryopreservation

1. Recovery of frozen cells

Quickly shake and thaw the cryovial containing 1mL of cell suspension in a 37 ℃ water bath, then add it to a centrifuge tube containing 4-6mL of complete culture medium and mix well. Centrifuge at 1000RPM for 3-5 minutes, discard the supernatant, and resuspend the cells in complete culture medium. Then add the cell suspension to a culture bottle (or dish) containing 6-8ml of complete 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 80% -90%, subculture can be carried out. For the passage of adherent cells, the following methods can be referred to:

2.1 Discard the culture supernatant and rinse the cells 1-2 times with PBS that does not contain calcium or magnesium ions.

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

2.3 Gently mix and aspirate, centrifuge at 1000 RPM for 3-5 minutes, discard the supernatant, add 1-2 mL of culture medium and blow evenly. Divide the cell suspension into new T25 bottles in a ratio of 1:2, add 6-8ml of new complete culture medium prepared according to the instructions to maintain the growth vitality of the cells, and subsequently subculture according to the actual situation in a ratio of 1:2-1:5.

3.Cell cryopreservation

After receiving the cells, it is recommended to freeze a batch of cell seeds during the first 3 generations of cultivation for subsequent experiments. Taking T25 bottles as an example below;

3.1 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 × 106~1 × 107 live cells/ml

3.2 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 × 106~1 × 107 live cells/ml per 1ml of cryopreservation solution. Label the cells with their names, generations, dates, and other information.

3.3 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.

Precautions

1. 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.

2. It is recommended to always use protective gloves, clothing, and a face mask when reviving frozen cells. Attention: The cryotube immersed in liquid nitrogen will leak and gradually fill with liquid nitrogen. When thawing, the conversion of liquid nitrogen into gas phase may cause the container to explode or the lid to be blown off with dangerous force, resulting in flying debris and causing personal injury.

3. All products provided by our company are for scientific research experiments only and are strictly prohibited from being used for clinical or other purposes.

1. Source: Mouse brain, microglia;

2. Morphology: Loose epithelial cell like adhesion, mixed growth of suspended and adherent cells;

3. Content:>1x106 cells;

4. Specification: packaged in T25 bottles or 1mL cryovials.

Bv2 cells are derived from the German collection of microorganisms and cell cultures (DSMZ) in Germany, derived from C57BL/6 mouse microglia, expressing nuclear v-myc and chromosomal v-raf oncogenes, and surface expressing envgp70 antigen. They exhibit phagocytic characteristics in morphology, phenotype, and function.