I am currently searching for suitable protocol of my study which related to macrophage isolation from lung and kidney.
The primary cell will then be subjected to flow cytometry, immunofluorescent as well as cell enrichment.
can anyone suggest good protocol either with enzyme or no enzyme usage.
thank you in advance.
Cell culture. Primary CKC were prepared from the kidneys of 1–2 week old Rhode Island Red chicks as previously described (Barrow & Lovell, 1989 R3 ). Briefly, kidneys were removed aseptically, teased apart and trypsinized with versene (0·8% NaCl, 0·02% KH 2PO 4, 0·15% Na 2HPO 4, 0·02% KCl and 0·02% EDTA). Cell concentrations were adjusted to 1×10 6 ml −1 in complete DMEM (Dulbecco’s modified Eagle’s medium) supplemented with 12·5% (v/v) heat-inactivated foetal bovine serum (FBS), 10% (v/v) tryptose phosphate broth (Difco), 25 U nystatin ml −1, 100 U penicillin ml −1 and 1 μg streptomycin ml −1 (P/S), at pH 7·0 and grown in 1 ml per well in 24-well Nunclon plates (Nunc) for 72 h at 37 °C, 5% CO 2. Two hours prior to use in invasion assays, the media was replaced with DMEM without antibiotics.
From one of my old papers. Don't have a method for lung sorry
So the method above is just for the kidney in general. I don't think you will get much yield of macrophages out of lung or kidney. Neither are that rich in the cell type. Whilst it is possible to grow monocytes from blood or better grow bone-marrow derived macrophages, getting many from other tissues will be tricky. You can use a simple trick in that macrophages are much more adherent than other cell types-we have used this to isolate splenic macrophages but have not cultured them for any length of time. The only issue is the presence of thrombocytes, though these survive poorly in culture.
thank you so much for your answer @Paul Wigley..
im not going to culture the macrophage.. im trying to use fresh tissue to extract the macrophage and then immediately process for the flow cytometry and proceed for cell enrichment.. do you think its ok?
You should get about an 80-90% MHC class II positive population. Thrombocytes are you enemy here. I would process tissue and leave 24h in a suitable media to lose the thrombocytes. Then wash and either trypinise or cold shock off the population. You can but try.
hi @paul wigley.. i try to culture my primary cell obtained and put in MEM supplemented with FBS and 1% pen/strep.. is it ok to use this media?
then i need to remove the macrophage adherence using trypsin or cold shock?
(cold shock means that i put in -20, and then take out.. is it?)
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