This experiment is mainly used for primary culture of adipocytes.
Operation method
Primary culture of adipocytes
Principle
The dispersed cells from the adipose tissue were filtered with a 25txm sieve to exclude mature adipocytes, and the filtered stromal-vascular (SV) cells were cultured in chemically-limited serum-free culture medium, which resulted in the dominant growth of preadipocytes and the gradual accumulation of fat to develop into mature adipocytes.
Materials and Instruments
Male Sprague-Dawley rats Move 1. Main experimental materials (1) Cell source: 4-week-old male Wistar or other strains of rats. (2) Wash solution: Ca2+ and Mg2+ free PBS with 100 U/mL penicillin and 100 μg/mL streptomycin. (3) Digestive solution: 2 mg/mL collagenase solution, DMEM culture solution preparation, and add bovine serum albumin 20 mg/mL. (4) Culture solution A: DMEM culture solution with 10% fetal bovine serum, 100 U/mL penicillin, and 50 μg/mL streptomycin. (5) Culture solution B: culture solution A with 33 μmol/L biotin and 17 μmol/L pantothenate. (6) Culture solution C: DMEM/Ham F12 (1:1, V/V) mixed culture solution with 15 mmol/L NaHCO3, 15 mmol/L HEPES, 33 μmol/L human biotin, 17 μmol/L pantothenate, 100 U/mL penicillin, 50 μg/mL streptomycin, pH 7.4. (7) ITT culture solution: culture solution C with 5 μg/mL insulin, 10 μg/mL transferrin, 200 pmol/L triiodothyronine (T3). (8) Sieve mesh: nylon sieve mesh with a pore size of 25 μm. 2. Methods of operation A. Sampling (1) 4-week-old male rats were anesthetized with ether and then killed by decapitation. (2) Cut the fat pad from around the epididymis under aseptic conditions, put it into a petri dish and remove the blood vessels as much as possible, and rinse with cleaning solution for 3 times. B. Isolation of cells (1)Sufficiently cut the tissue, put it into the digestive solution, and digest it by oscillation in 37℃ water bath for 40min. (2) Pour the digestive solution into the beaker with culture solution A, blow repeatedly with a pipette, and use a pore size of 25μm The filtrate and unfiltered tissue pieces were collected by filtering through a nylon sieve with a pore size of 25 μm. (3)Centrifuge the filtrate at 1800r/rain for 5min, discard the supernatant; add culture medium B to make SV cell suspension, and store it temporarily in the refrigerator at 4℃. (4) Repeat the treatment with unfiltered tissue block once, and centrifuge the precipitated SV cell mass with culture solution B to make the SV cell suspension. (5) Combine the SV cell suspensions obtained twice and mix by pipetting and blowing; take a small amount of SV cell suspension, count, and adjust the cell density. Count without counting the blood cells. C. Primary culture (1) Inoculate the cells at a density of 104 cells/cm2 in a Petri dish with culture medium B. Cultivate the cells in a 37℃, 5% CO2 incubator for 12～24h. (2) After cell apposition, culture medium C was washed twice, followed by addition of culture medium C for 1h of transition culture. (3) Discard culture solution C, and wash with PBS for 2 times. (4) ITT culture medium was maintained in culture and changed every 3 days. Caveat 1. Adipocytes can be harvested either from around the epididymis or subcutaneously from the rat retroperitoneum or inguinal area; if harvested from the inguinal area, the udder tissue needs to be removed. 2. ITT culture medium is a chemically limited serum-free medium for the conversion of rat preadipocytes to adipocytes and cannot induce SV cell proliferation, but culture medium B can. 3. Culture medium B (containing serum) pre-culture SV cells for 12~24h is to promote cell attachment, if the growth matrix components (such as fibronectin, laminin, collagen, etc.) that can promote cell adhesion are used to pre-coat the culture surface, pre-culture can be dispensed with, and the culture surface can be pre-cultured. Common Problems Source Commonly Used Medical Cell Cultures For more product details, please visit Aladdin Scientific website.
KRBH buffer KRBH-A buffer DMEM-A DMEM-B Collagenase
Petri Petri dishes Conical centrifuge tubes Polypropylene tubes Low-density polypropylene bottles Nylon filters Pointed dissecting scissors Perry forceps Plastic cases Guillotines Pipettes