Hi, I am currently trying to isolate immune cells from mouse colon tissue for my project. For this purpose, I am using a protocol which includes ;
- Surgical removal of the colon tissue from mouse - Cleaning the tissue from surrounding fat and feces - Cutting the colon longitudinally and into small pieces (~4, 5 mm) - Incubating with 2 mM EDTA at 37C with mild shaking for 15 min x2 (removal of EDTA in between) - Washing the pieces in a strainer with 1X PBS x4 - Cutting the colon tissue into smaller pieces with a scissor - Digestion for 1 hour at 37C with digestion mix (1mg/mL Collegenase II, 2U/mL Dispase II, 80ug/mL DNAse I in DMEM with Glutamax) - 20 sec vortex - Quenching the rxn with 2 mM EDTA and washing with PBS/2%FCS - 10 sec vortex - Filtration (40 um) into new falcon to remove tissue - Centrifugation for 7 min, 1500 rpm.
At the end of this protocol, I always encounter formation of a slimy, white, powder like precipitate in some of my samples. These samples take quite some time to filter through 40 um filters and at the end, usually no cell pellet was observed in these tubes. I have previously tried to optimize the protocol by changing the concentration of digestion mix, digestion time, EDTA incubation time and washing the tissues after EDTA incubation with several ways, however none of my attempts stopped the formation of this precipitate. It appears to be happening randomly between my samples in every experiment irrespective of the genotype, treatment etc.
I couldn´t be able to pinpoint the source of error in my procedure, so my intention is to get the opinion of you, fellow scientists. Is there any other people present in this platform that also works with a similar protocol and maybe encounter similar problems? I am open to further discussion and suggestions.
I can give you my opinion, although I am not an expert in the field. It can't be both slimy and powder... If its slimy my best guess is DNA so try to increase the DNase. If its powder, perhaps its calcium salts, so you can increase the EDTA or add a salt- cell cleaning solution. Do you have o picture of the precipitate?
- Badges
- Science topic
- Similar topics
- Analytical Chemistry
- Extraction