I'm currently working on isolation of dendritic cells from the murine colon, but haven't been able to get too much. I'm currently using EDTA washes followed by a collagenase/DNAse digestion, then using a Percoll gradient (although I have been using a pelleting method that's working a little better). When ran through the flow, it shows I have cells, but nothing is staining the way it should. I'm going to be doing a viability stain to see if these are live cells in the coming week. Does anyone have any advice on how I should proceed?
Thanks!
I would check your method against published protocols to see whether the reagent concentrations you are using and duration of your processing steps are roughly in-line with those employed by others. I would guess that the Percoll step doesn't help much and probably loses a lot of cells... Viability staining and looking at the cells on a slide will probably help to work out what is going on. I assume that you are chopping up the tissue into small pieces before processing?
Maybe try this for reference; http://onlinelibrary.wiley.com/doi/10.1002/1521-4141%28200205%2932:5%3C1445::AID-IMMU1445%3E3.0.CO;2-E/full
Agree with Neil. I generally add Dispase to the enzymatic digestion step and use a 40%/80% Perccoll gradient to obtain lymphocytes and monocytes. Make sure everything you do after removing the tissue is ice cold and add a fixable viability dye as you mentioned to make sure you're not killing the cells. Typically you can expect 5-10*10^6 cells/small intestine depending on the microflora in your strains. See Ivanov 2008, Cell Host and Microbe for details. Good luck.
Some issues you may want to address to improve yield and viabilitu. 1. Check that your HBSS or PBS you are using in wash with EDTA does not have ca2+ or Mg2+. 2. Ensure you are washing thorougly your intestinal pieces after EDTA treatment (inhibits collagenase). 3. ensure you are using an ACTIVE dose units of collagenase as batches vary even from same source. 4. Use medium like RPMI without mercaptoethanol (inhibits collagenase). 5. Use cocktail of protease inhibitor with DNAse. 6. Percol is necessary to remove dead cells because they release DNA which causes clumping of cells. 6. You do not need to do more than one/two digestions with collagenase. Further digestions affect viability of cells. Check viability of your cells. 7. Use MACS beads to isolate DCs.
Thank you everyone for the responses! I do cut up the colon into small pieces before continuing on. This protocol I have advised to incubate at room temperature, but I will try it on ice. As for the EDTA washes, I wasn't washing the colon in between so I am definitely going to try that. Thank you again for the advice! I have been struggling with this for a couple weeks now.
Also, Toufic, I had originally tried Percoll without a ton of luck. I can't locate the "band" of lymphocytes between me 30%/100% gradient. Is there a better combination to use? Specifically for dendritic cells?
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