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Flow cytometry of murine blood
J. Orthel.  6/27/03
mf revised 9/04
  Lineage Analysis of Blood

Materials:
Capillary tubes
1.5 mL Eppendorf microfuge tubes
15 mL conical centrifuge tubes
96-well V-bottom plates (Corning Costar 3894, from Fisher)
Flow tubes (Falcon 352054)

Reagents:
0.5 M EDTA pH 8.0
PBS + 0.5% heat inactivated FBS
RBC Lysis Buffer
    4.15 g NH4Cl
    0.5 g NaHCO3
    0.0186 g Disodium EDTA
    200 mL H2O

Antibodies:
     [Final]             Catalog #
Fc Block (2.4G2)   
 1:1000
 Pharmingen 553142
FITC-IgG2b Isotype 
1:100 Pharmingen 553988
PE-IgG2a Isotype   1:100 Pharmingen 553930
Mac-1 (CD11b)-IgG2b-FITC 
1:100 Pharmingen 553310
Gr-1(Ly6G)-IgG2b-FITC 
1:100 Pharmingen 553127
CD3-IgG2b-FITC   1:100 Pharmingen 555274
LY5a (CD45.2)-Biotin (recognizes C57)
 1:100 Pharmingen 553771
Ly5b (CD45.1)-PE  (recognizes SJL)
 1:100 Pharmingen 553776
Streptavidin-TriColor (SATC)
 1:100
 Caltag SA1006

Procedure:
  1. Collect 200 - 500 uL blood from each mouse.  Mix with 50 uL 0.5M EDTA in 1.5 mL eppendorf tubes.
  2. Add 250 uL blood to 5 mL RBC Lysis Buffer (20x vol. blood) in 15 mL conical tubes.
  3. Spin down white cells 1500 RPM x2 min.
  4. Aspirate lysate and wash by resuspending cells in 5 mL PBS/FBS. 
  5. Spin down at 1500 RPM x2 min.
  6. Resuspending cells in 500 uL PBS/FBS with 1 uL Fc Block (1/500)
  7. Add 150 uL to each well of a 96 well V-bottom dish.
  8. Add 50 uL 1º Ab Master Mix (the mix is a 1/25 dilution of each 1º Ab in PBS/FBS).
  9. Include 1 well with a combination of Isotype controls for setting voltage.  Also include 1 well for each of the 1º Ab as single positive controls for setting compensation.  
  10. Incubate 60 min at 4ºC. 
  11. Spin 1500 RPM x2 min.  Discard supernatant by shaking it out once into the sink, and blot inverted plate on paper towel. 
  12. Wash by adding 200 uL PBS/FBS to each well, and mix by pipetting up and down. 
  13. Immediately spin at 1500 RPM x2 min. and discard supernatant.
  14. Add 150 uL of 2º Ab (i.e. Streptavidin-TC) Master Mix.
  15. Incubate 30 min at 4ºC, then spin at 1500 RPM x5 min.  Shake out supernatant.
  16. Resuspend in 200 - 500 uL of PBS/FBS and transfer to 5 mL flow tubes.
  17. See the flow cytometry protocol for 3-color flow.

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