I realize that the work flow in most flow-based studies of neutrophils is such that the cells are analyzed without ever becoming adherent to the cell culture surface, ie, as soon after isolation as possible.
However, I am interested in looking at flow markers post ex vivo stimulation (and thus post adherence) as my research relates to NETosis. I am worried about using physical or trypsin based detachment methods due to the possibility of cell rupture or loss of antibody binding to markers of interest. I am considering Accutase, DNAse I, or TryplE for this purpose- which would you consider the most promising, if any?
Sounds like accutase + dnase would target the two problems you have in theory -- ECM attachment and DNA nets. I think those are good ideas. It does not seem like they tell you what's in tryple. So it's hard to speculate about that.
Since all of that is very cheap, you should try everything alone and in combination. Include trypsin too. Experimental testing beats theorizing every hour of the day. You can't afford overlooking the best solution because of some theoretical idea. Try it, and then you'll know.
Sounds like accutase + dnase would target the two problems you have in theory -- ECM attachment and DNA nets. I think those are good ideas. It does not seem like they tell you what's in tryple. So it's hard to speculate about that.
Since all of that is very cheap, you should try everything alone and in combination. Include trypsin too. Experimental testing beats theorizing every hour of the day. You can't afford overlooking the best solution because of some theoretical idea. Try it, and then you'll know.
Hello,
Not sure whether this works for neutrophils too, but it certainly works for monocytes which also adere to culture plastics during ex-vivo experiments in cultures. It is a "trick" used by technicians. You van try detaching your cells by exposure to cold PBS with EDTA. As far as i know, this is the least agressive method for detaching peripheral blood cells from culture plastics.
Also, as John mentioned above, you should decide on a couple of methods and test them in a series of mini-experiments. Very well said: "experimental testing beats theorizing every hour of the day" .
We should make a quote out of this.
https://www.researchgate.net/post/Whats_the_best_way_to_detach_primary_macrophages_from_surface_of_tissue_culture_plates_dishes
Very interesting comments to this question, you may wanna take a look.
Not much info about neutrophiles being available around :-/
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