Primary Cell Culture: Isolation of Rat Peritoneal Macrophages
Advantages of Primary Cell Culture
Primary culture is the culture of cells directly obtained from animal tissues.
It has the following advantages:Â
More in vivo like characteristics
Cells can be obtained from rare or unusual species
Patient derived tissues can be utilized for direct experimentation
Cells that are not available as cell lines
About Rat Peritoneal Macrophages Cell Culture
The two most convenient sources of primary murine macrophages are the bone marrow and the peritoneal cavity.
Resident peritoneal macrophages can readily be harvested from mice and purified by adherence to tissue culture plastic.
The injection of thioglycollate broth into the peritoneal cavity produces an inflammatory response allowing the purification of large numbers of elicited macrophages.
The macrophages obtained by the following protocol are normally 90% pure and can be used further for other assays like drug uptake, phagocytosis etc.
Cell Culture Requirements
Sterile:
1. Phosphate Buffer Saline (PBS) (i.e. 137mM NaCl, 2.7mM KCl, 10mM Na2HPO4.2H2O and 2 mM KH2PO4, pH 7.4).
2. Geys solution: Stock A (0.65M NH4Cl, 0.024 M KCl, 10.56 mM Na2HPO4, 0.89 KH2PO4, 0.02 M Glucose, 0.141 mM Phenol Red); Stock B (0.207 mM MgCl2.6H2O, 0.057 mM MgSO4.7H2O, 0.306 mM CaCl2); Stock C (2.68 mM NaHCO3); Geys Solution (Make Fresh): 20 Parts of Stock A 5 Parts of Stock B 5 Parts of Stock C 70 Parts of autoclaved deionized water.
3. RPMI medium supplemented with 10% FBS and antibiotics
4. FBS
5. 23G and 18G needles
6. 10ml syringe
7. 3% w/v Brewer’s complete thioglycollate broth: 3g dissolved in water, Autoclave and store in dark and should be allowed to age for atleast 15 days before using for the experiment
8. Scissors and forceps
Non-sterile:
9. 70% v/v isopropyl alcohol or ethanol
10. Cotton
11. Ice
Cell Culture Procedure
1. Inject the rat with 5 ml aged thioglycolate medium in the peritoneal cavity using a syringe with 27 G needle.
2. After 72 hr and immediately before harvesting the macrophages, sacrifice the rat by CO2 asphyxiation.
3. Disinfect all the external areas of the rat with 70% IPA.
4. Make a small incision in the abdominal region and gently rip the skin downward to expose the intra-peritoneal cavity.
5. Inject 10 ml of sterile PBS into the intra-peritoneal cavity using a syringe with a 23 G needle and massage the abdomen gently for 2 min.
6. Retract the injected PBS using a syringe with 18 G needle. Care should be taken not to injure the internal organs during this process.
7. Centrifuge the Macrophage suspension at 1300 rpm, 7 min and 40C and observe the pellet.
Note: If RBCs are visualized, then add 1ml of complete Gey’s solution and incubate on ice for 2-5 minutes. Gently overlay 1ml of FBS to this suspension and spin at 300g for 10 minutes at 4oC. Wash once with PBS containing 5% FBS and then resuspend in 1 ml of RPMI supplemented with FCS and antibiotics.
8. Count the cells using hemocytometer
9. Plate the resident peritoneal cells RPMI culture medium at 3 x 10^5 cells per well in a 24-well tissue culture plate or 106 cells in 30mm sterile petridish and incubate for 60 mins at 37°C.
10. Remove the nonadherent cells by washing 3 times in 500ml warm PBS, using a gentle swirling action.
11. The adherent cells normally contain >90% macrophages
12. These macrophages will be used for drug uptake assay
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