Using an enzymatic digestion method(Collagenase treatment) i am not able to derive single cell suspension.The cells come out as clumps and if try to pass through the 40 micron filter i am getting very few cells.Is there any method subsequent to collagenase treatment that could give me large number of single cells?
I am interested in sorting cancer stem cells directly from tumor tissue without establishing a primary cultures.
Dear Dr. K. Padmanahan
We prepare single nuclei suspensions from formaline fixed parafin embedded tumor tissue by a modification of the method described by Hedley et al in our work with success.
J Histochem Cytochem. 1983 Nov;31(11):1333-5.
Method for analysis of cellular DNA content of paraffin-embedded pathological material using flow cytometry.
Hedley DW, Friedlander ML, Taylor IW, Rugg CA, Musgrove EA.
Abstract
A method has been developed that allows flow cytometry to be used for measuring the cellular DNA content of paraffin-embedded human tumors. Thick (i.e., 30 micron) sections were cut from tissue blocks using a microtome and dewaxed in xylene. The sections were then rehydrated by sequentially immersing them in 100, 95, 70, and 50% ethanol before finally washing in distilled water. Single cell suspensions were then prepared by incubation in 0.5% pepsin, pH 1.5, at 37 degrees C for 30 min. The cells were counted, washed, and stained with 1 microgram/ml 4',6'-diamidino-2-phenylindole for 30 min, and DNA content was measured using an ICP 22 flow cytometer. There was a good correlation between the DNA histograms produced using this method and those obtained using unfixed tissue from the same tumor stained with ethidium bromide plus mithramycin. This method allows the retrospective study of archival material where the clinical outcome is already known, and it should, therefore, be particularly useful for determining the prognostic significance of abnormal DNA content measured by flow cytometry.
Hope this method could be usefull for your experiments.
All the best
Wanja Kildal
I have isolated primary neurons from rat brain. To get individual cells, I used 0.25% Trypsin at 37C for 30 min. Add DNAse, BSA and Trypsin inhibitor to the trypsinized cels and triturate the tissue peices using reducing orifice tips like 1 ml tip attached with 200 ul then to a 10 ul tip for 20 times towards the tube wall. Be careful not to produce bubbles in the media, this will reduce the cell viability. If you need more details on the concentration of trypsin inhibitor trituration solution, please let me know.
Thanks a lot for all your answers i will try out these suggestions and let me see what happens
Thanks everybody a combination of all solutions were really helpful got single cell suspension of good viability.Thanks a lot.
Dear Krishnanand,
I request you to share your optimized protocol because I am also looking for similar kind of experiment and its becoming to hard to get a single cell suspension. I am using 1% trypsin in PBS but I am getting dead cells. I would request you to share it either on RG or you can also mail me for that I will be thankful to you
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