Haemocytometer cell counting

[Peptides]

Hello all, I have a question regarding a recent practical I performed using a hemocytometer during my Immunology class.

 

During this practical I dissected out a mouse spleen, to which I added PBS. A 10ul sample of this suspension was then mixed with an equal volume of trypan blue and loaded into the haemocytometer chamber. My cell count was as follows: White cells: 203, Blue cells: 42.

 

100ul of the original spleen suspension was then added to 1ml red blood cell lysis buffer. As this is a hypotonic solution which lyses red blood cells, I expected to see more dead cells. However, when mixed with trypan blue and loaded into the haemocytometer chamber, I saw no cells at all. I tried repeating this section of the protocol but again observed no cells.

 

Why might this have been? I'm guessing that maybe all the cells were red blood cells and therefore lysed, but then wouldn't I have at least seen some blue cells, definitely more than before lysis? If I did happen to obtain a cell count after lysis, but the total count was different to that obtained before lysis, could this just be a result of the different samples of spleen suspension taken?

 

Lastly I'd like to calculate the number of red and white cells in the spleen. To do this, I'm assuming I would take the difference between the blue cell count before lysis and the blue cell count after lysis to be the number of red blood cells in the sample. The white cell count would then be the white cell count after lysis. Does this sound correct? Also, I'm confused on and really have no idea about how I would then use this to calculate the number of red and white cells in the spleen

 

Any help on this would be much appreciated.