Lineage Analysis of Blood
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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)
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 NH4 Cl
0.5 g NaHCO3
0.0186 g Disodium EDTA
200 mL H2 O
PBS + 0.5% heat inactivated FBS
RBC Lysis Buffer
4.15 g NH4 Cl
0.5 g NaHCO3
0.0186 g Disodium EDTA
200 mL H2 O
|
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:
- Collect 200 - 500 uL blood from each mouse. Mix with 50 uL 0.5M EDTA in 1.5 mL eppendorf tubes.
- Add 250 uL blood to 5 mL RBC Lysis Buffer (20x vol. blood) in 15 mL conical tubes.
- Spin down white cells 1500 RPM x2 min.
- Aspirate lysate and wash by resuspending cells in 5 mL PBS/FBS.
- Spin down at 1500 RPM x2 min.
- Resuspending cells in 500 uL PBS/FBS with 1 uL Fc Block (1/500)
- Add 150 uL to each well of a 96 well V-bottom dish.
- Add 50 uL 1º Ab Master Mix (the mix is a 1/25 dilution of each 1º Ab in PBS/FBS).
- 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.
- Incubate 60 min at 4ºC.
- Spin 1500 RPM x2 min. Discard supernatant by shaking it out once into the sink, and blot inverted plate on paper towel.
- Wash by adding 200 uL PBS/FBS to each well, and mix by pipetting up and down.
- Immediately spin at 1500 RPM x2 min. and discard supernatant.
- Add 150 uL of 2º Ab (i.e. Streptavidin-TC) Master Mix.
- Incubate 30 min at 4ºC, then spin at 1500 RPM x5 min. Shake out supernatant.
- Resuspend in 200 - 500 uL of PBS/FBS and transfer to 5 mL flow tubes.
- See the flow cytometry protocol for 3-color flow.
