Hello
Have any one tried using the EDTA solution instead of trypsin in detach the adherent cell, in flow cytometry assay, particularly in High throughput analysis
Regards
Hi Asma,
It all depends on how sticky are your cells. Typically for HEK cells it works just fine - we use Versene (EDTA based solution) 10 min at 37oC and then gentle pipeting and they all detach well. But if your cell line is really sticky then it becomes much more difficult to detach cells without damaging them.
we have used Accutase from Sigma. It is EDTA-based (0.5mM) but also has proprietary enzymes of undeclared origin!! so it may not be useful for some applications. For our purposes, it removed sticky cells effectively and rapidly without apparently affecting cell surface protein expression and function.
http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Datasheet/6/a6964dat.pdf
I've found that this has led to huge amount of cell death in lines that I've tried this in. As mentioned already, more strongly adherent lines respond poorly to this method
Thanks all for kind reply
Actually I would like to minimize the washing and centrefugation steps, which are affecting my cell and reading as well
I am working with MCF-7 and LnCaP cell line
What about Accutase solution, is there any one work with these tow cell lines and how it is work
I am Working on 96 well plate , i want to minimize these steps as possible
How to dissociate organoid cultureI to get single cell suspension for Flowcytometry application? Trypsin treatment is affecting surface marker expression.
I often need to dissociate mouse organs (lymph nodes, spleen) into single cell suspensions. To do this, I use very well established protocols that essentially are just mashing your cells through a 40 micron nylon filter with a syringe plunger, washing the membrane with buffer, and iteratively doing this a few times. Works beuatifully. Depending on your cell type, you might want to try this for your organoids.
https://www.youtube.com/watch?v=N0jftyYqM38
Safe to use collagenase and dispase for organoids too. However, it is also depending on origin tissue type of your organoids. Some tissues are very rigid and tight e.g. breast cancer tissues, some are a bit loose and delicate. So have to adjust according to the type of tissues. You can also use trypsin, collagenase alone. You can assist mechanical dissociation at the same time.
Regarding the detachment of cells is highly depending on type of cells too. Some cells easily detach even with PBS or EDTA alone or just gently knock the flask or lower % of Trypsin alone, Trpsin+EDTA, 5% 2.5% 1.25% 0.5% Trpsin+ 0.5mM EDTA or TripLE or Accutase etc. Just used whatever way works for you.
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