J. Orthel. 6/27/03
mf revised 9/04
Lineage Analysis of Bloodmf revised 9/04
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 NH4Cl
0.5 g NaHCO3
0.0186 g Disodium EDTA
200 mL H2O
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:
- 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.
