Measuring Cell Size Using Flow?

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beths
beths's picture
Measuring Cell Size Using Flow?

Hi All,

I'm trying to measure cell size using flow. It's simple enough but I find when I use a hypotonic solution, my cells appear to be shrinking!! I grow my cells in submerged culture and split them from the flask. Once I get a cell pellet I resuspend this in a bath solution of 140mM NaCl, 2.5mM KCl, 1.2mM CaCl2, 0.5mM MgCl2, 5mM Glucose and 10mM HEPES. The hypotonic contains 75mM NaCl.
 
Any ideas what's going wrong or any suggestions as to how to do it differently?

Many Thanks

x

Rajeshwari patel
Rajeshwari patel's picture
dear beths,

dear beths,

I am not use to with the flow cytometer  but from my cell culture experties i can say the idf you resuspend your cells in the PBS  your problem will solve

thanks
rajeshwari

dav0082
dav0082's picture
Hi beths

Hi beths
whenever you perform flow cytometric analysis ideally your buffer should be ca and mg  free to avoid cell clumping.
I generally use the PBS i.e. ca and mg free
                                               

marcus muench
marcus muench's picture
 Another technique to avoid

 Another technique to avoid the effects of clumps is to use doublet discrimination if your flow cytometer supports this.  For instance, on an LSRII, if you set the machine to record FSC height, area and width then you can plot these parameters against each other.  Single cells should form a straight diagonal  whereas doublets tend to have ahigher forward scatter in one parameter than the other (they are off the diagonal).