Are these cells still viable? (Trypan Blue Stain) - Using this stain for the first time against Sf9 cells (Sep/09/2008 )
Hello. I am using 0.4% Trypan Blue stain to check for viability of my insect cells. To perform the staining, i mix 100 ul of the cells with 100ul Trypan blue 0.4%. When i visualize the cells under microscrope, all cells stain blue!
I am thinking whether the stain to cell ratio that i use is too high? Or I cannot differentiate between viable and non-viable cells? Or, the cells are really rest in peaces?
The photo is attached below. Thanks for the reply. 
I am thinking whether the stain to cell ratio that i use is too high? Or I cannot differentiate between viable and non-viable cells? Or, the cells are really rest in peaces?
The photo is attached below. Thanks for the reply.
I have no experience with insect cells but in my opinion, your cells look dead
best way to find out is to split them anyway and see if they recover and expand, but I would also start up a new batch 
I am thinking whether the stain to cell ratio that i use is too high? Or I cannot differentiate between viable and non-viable cells? Or, the cells are really rest in peaces?
The photo is attached below. Thanks for the reply.
Dear dcch,
I am afraid that all your cells are dead. The EXCLUSION assay " Trypan blue" is just that
Cells that exclude the dye are alive
Your cells DO NOT exclude the dye so they are dead.
I hope these were not your last vial?
Regards
Rhombus
These are the floating cells which did not adhere, so i did the staining to check whether the cells are REALLY non-viable or they are just healthy but did not want adhere.
In my planning, if they are healthy, i will just wash away the DMSO and put into suspension culture. Seems i do not have to to do this additional step anymore.
To Rhombus: Yes this is the last vial. The remaining cells which adhere to the flask is low in number. I will check back the adherence tomorrow. Hopefully everything is fine.
Is 1:2 (1 volume of cell to 1 volume of Trypan blue 0.4%) stain dilution reasonable?
I read somewhere that they use 1:10, but mostly they stick to 1:2.
I read somewhere that they use 1:10, but mostly they stick to 1:2.
It should be 1:2 dilution, always has been, always will be
Rhombus
Thanks for the reply Rhombus!
Actually i read about 1:10 in Invitrogen manual which sounds below:
7.3 (i. e. phosphate-buffered saline).
3. Add 0.1 ml of trypan blue stock to 1 ml of cells.
4. Load a hemacytometer and examine immediately under a microscope at low
magnification.
5. Count the number of blue staining cells and the number of total cells.
Wondering why they suggest this...
Anyway i accept 1:2 dilution and really appreciate the replies above.