Primary culture of adipocytes
Primary culture of adipocytes
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