I am looking for a method of forming single cell suspension from tumor and normal tissue.
I went through some of the discussion (I got 2 only) and I found they have used 1% trypsin for 16 hour at 4 C. I did try to do the same but I am getting dead cells.
Because I want to fix the cells after forming single cell suspension so it is not working for me.
If, any body is familiar with this kind of work then please share your views with me.
I will be very much thankful to you.
I would like to serve any further information if required.
You may wish to mince the pieces by hand then digest for short periods of time until you are happy with the viability. (There are expensive machines and consumables for automated, reproducible processes).
There are likely more things to consider depending upon your downstream application.
I used the enzyme dispase to generate single cells from tissue. Pass your preparation through a cell sieve (45um) to remove remaining clumps.
Hi Amit
Since I have experience in digesting normal tissues, this what I can suggest. It may also work with tumor.
incubate your tissues with enzyme cocktail. dissect and mince tissue with sterile scalpel and incubate with 1 ml dispase+ 2 µg/ml collagenase B +0,001 % (w/v) DNAse in DMEM for 15 min at 37 °C (or more 30 min) or on shaker plate for 10 min. better digestion is the one at 37 °C. The resulting cell suspension will be filtered through 100 µm and 40 µm or 70 µm cell strainers (depending on the size or your cells of interest) and centrifuged at 400 x g, 5 min at 4 °C. optional, cells can be resuspended in cell blood lysis buffer (0.15 M NH4Cl, 10 mM KHCO3, 0.1 mM EDTA) for 5 min, to eliminate blood cells and washed in PBS and resuspended in a buffer and this is your cell suspension
best wishes
Thanks Every one for your valuable suggestion.....I will try to optimize my protocol with help of above given suggestions.............Thanks all
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