[Cytometry] Flow Cytometry on mouse bone marrow and blood

Abdul Khan khan.abdulh at googlemail.com
Mon Jul 13 16:02:00 EDT 2009


Dear All,

I am going to do flow cytometry on mouse bone marrow and blood to analyse
the monocytes and neutrophils. I would appreciate if you guys could help
me to fine tune my protocol with all your invaluable input. I have done this
on human peripheral blood but never done on mouse, so your help would be
very helpful. The protocol I have got so far is below:

1. collect the blood in Lithium-Heparin tubes

   - *Is there any specific Li-Heparin tube I should be using for collecting
   the mouse blood or I can use the same tube (6ml VACUETTE tube) I used for
   collecting the human blood samples ? What would you recommend to minimise
   the blood clotting as there would be around an hour delay before processing
   the samples.*
   - *What would be the best way to collect the bone marrow ?*

2. Lyse the RBCs with lysis buffer

3. Wash in staining buffer (can I use the same staining buffer for both
blood and bone marrow, please see below. Also, can someone tell me what is
main difference between the two staining buffers? I have got this from
published papers)

   - *For blood: PBS + 1% FBS + 0.09% Sodium Azide*
   - *For bone marrow: HBSS + 1% FBS + 10mM HEPES *

4. Incubate with mouse Fc blocker (CD16/CD32)

5. Incubate with fluorescent labelled antibodies

   - Do we usually dilute the antibody in staining buffer and add the small
   aliquots (10ul per 100ul of sample) ? I didn't dilute the anti-human
   antibodies in my previous experiments.

6. Wash twice in staining buffer

7. Read on FACS Calibur

   - Can I use the template I made for human blood as a starting point and
   do all the adjustment and compensation ?


The above protocol I have written down is all I got after reading published
papers. I have done the FACS on human whole blood but the protocol I used
was slightly different than this one - first I incubated the whole blood
with fluorescent labelled antibodies and then lysed the RBCs with lysis
buffer followed by washing and reading on FACS Calibur. Can I use the same
protocol for mouse blood and bone marrow ?

I would be very grateful for any help in this regard. Looking forward to see
your comments. Many thanks.


Regards,

Abdul Khan
University of Glasgow
U.K.


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