Microscope Settings - Microscope Settings for Trypan Blue Exclusion Assay (May/08/2008 )
I am performing mammalian lymphoblastoid cell line and peripheral blood mononuclear cell viability assays using Trypan Blue exclusion under an inverted phase-contrast microscope. I am a Molecular Biologist and not really accustomed to Cell Biology. I am having trouble fine-tunning the microscope for adequate dead cell:live cell counts. Right, on with the questions:
1.- Am I perhaps being too naive at expecting dead cells to be bloated, very blue and scarce...In other words "in your face"? At the time I encounter occasional big, light blue (eosinophil-like) not-very-round cells which are quite nice (text book dead cells, I think) but also encounter lots of small PBMCs of a darker hue. Are these small leukocytes also dead or just the result of refraction?
2.- Should I be using the Microscope as a brightfield or phase-contrast to read my hemocytometer and do trypan blue exclusion assays?
3.- Would anyone happen to have a decent english-written inverted phase-microscope manual (any make) that will help me fine tune my instrument, I didn't get one with mine and have not found anything on the web? By the way my Microscope is Chineese, so don't even go there...optics seem to be superb though!
4.- I also seem to have a bad case of bacteria or yeast-like objects (ca 3-5 microns in size) surrounding my PBMCs. This is unexpected as my cells have not been cultured, plastics are all new and sterile and the samples have only been subjected to Ficoll 1077 isolation. Any hints? Has anyone heard of yeast/bacteria living out of Ficoll?
5.- Would anyone of you experienced cell biologist be interested in moving to San Luis Potosí Mexico?
I use the hemocytometer and usually mix 90 microliter of trypan Blue to 10 microliters of Cell suspension, I use the x20 objective with phase contrast and in case of trouble clean thoroughlly the chamber with a bit of HCl (followed by extensive rinses with DD H20)
Most of the time the dead cells appears light to dark blue, he erythrocyte however are darker than other living cells (specially B cells) but are not blue at all so you should be able to do the differences
In case you really need an accurate measurement use PI staining and FACS analysis (or even the Annexin V Apoptosis staining kit from Roche)