I read some papers in which they used sigma C2139 (which is collagenase VIII) while some papers reported colagenase III. Can I use any collagenase to isolate interstitial cells for flow cytometry or is there a specific type of collagenase that should be used? It'd be great if you could share the protocol with me.
well.. I used collagenase IV together with DNase. It works fine for FACS.
Thanx Agrawal. can you share your protocol. because my colleague was saying its may depends on protoclol methodology or different reagent use. it would be helpful for me.
1. Capture lung (after perfusion with PBS) tissue and keep at 4 ⁰C. Chop it with scissor/blade to make a fine paste.
2. Collect the paste in a 50 ml falcon tube using 2% RPMI media. Centrifuge at 2000 rpm at 4⁰C for 10 min to collect the tissue as pellet.
3. Resuspend the tissue in 1 mg/ml collagenase IV solution in 10% RPMI supplemented with 0.01 mg/ml DNase and let the digestion proceed at 37⁰C for 20 min.
4. Strain the digested paste through 40 µm cell strainer and centrifuge the flow through at 2000 rpm for 10 min at 4⁰C.
5. Resuspend the pellet in 5 ml of 2% RPMI solution.
6. Count total no. of cells using hematocytometer. Mix 5 µl of cell suspension with 45 µl of trypan blue (to identify live cells) and count.
7. Proceed for FACS
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