Staining and Flow Cytometry for BLT mice

Dong Sung An’s Lab (UCLA)

12/13/19 by Nai

In brief:
Staining protocol: 100 uL of mouse blood samples or 1 million cells of isolated mouse bone marrows were stained with 50 uL of master mixed of 1 ul of each following monoclonal antibody to human CD45-eFluor 450 (HI30:eBiosciences), CD3-APC H7 (SK7:BD Pharmingen), CD4-APC(OKT4:eBiosciences), and CD8-PerCP Cy5.5 (SK1:BioLegend), and CD19-Brilliant Violet 605 (HIB19: Biolegend), incubated for 30 min at room temp and covered with foil. After that they were added 1 mL of 1x RBC lysis buffer (RBCL; 4.15 g of NH4Cl, 0.5 g of KHCO3, and 0.019 g of EDTA in 500 mL of H2O) and incubated for 1 min at room temp, spun down at 2,400 rpm x 3 min, and removed supernatant, repeatedly this step for 2 times. Then the samples were washed with 1 mL of FACS
buffer (2% fetal calf serum in phosphate-buffered saline [PBS]), spun down at 2,400 rpm x 3 min, removed supernatant, and added 0.3 ml of fix buffer (1% formaldehyde in PBS) and examined with Fortessa flow cytometers (BD Biosciences). The data were analyzed by FlowJo V10 (TreeStar) software.

<Reagents>
Red Blood Cell Lysis Buffer– 10X (store at cold room)
Ammonium Chloride (NH₄Cl) 82.9 g
Potassium Bicarbonate (KHCO₃) 10.0 g
Ethylenediamine tetraacetic acid (EDTA) disodium salt 0.37 g
Water, glass distilled 1.0 liter
After filtration through 0.22 µm, store at 4C.

1x Red Blood Cell Lysis Buffer
Diluted 10X RBC buffer with ddH₂O and filtration, store at 4C.
Note: buffer should be used at room temp.

FACS Buffer: 2% FCS in PBS (store at 4C)
Add 10 mL of FCS into 500 mL of 1 xPBS (cellgro: cat#21-022-cv).
Fix Buffer: 1% formaldehyde in PBS (store at 4C)
Add 14 mL of 37% Formaldehyde (37% formaldehyde solution: Fisher: # F79-500) into 500 mL of 1xPBS (cellgro: cat#21-022-cv).
BD^TM CompBead: (BD cat#552843)

Antibody Master Mix1
1 ul of anti-human CD45-eFluore 450 (HI30:eBioscience; #48-0459-42)
1 ul of anti-human CD3-APCH7 (SK7:BD Pharmingen: #560176 )
1 ul of anti-human CD4-APC (OKT4:eBiosciences:#17-0048)
1 ul of anti-human CD8-PerCPCy5.5 (SK1:BioLegend: # 344710 )
1 ul of anti-human CD19-BV570 (HIB19: BioLegend:#302235)
+ 50 ul of FACS Buffer/mouse sample.

<single staining>
1. Human PBMC 1x10⁶ cells unstained cells
2. Human PBMC 0.5x10⁶ cells + master mix1
3. 1 drop of positive CompBeads
a. + 1ul anti human CD45-eFluore 450 [PacBlue](eBioscience; #48-0459-41)
b. + 1ul CD3-APCH7
c. + 1ul CD4-APC
d. + 1ul CD8-PerCPCy5.5
e. + 1ul anti human CD19-BV605
f. Non
4. After staining cells, add 0.5x10⁶ cells of unstained PBMC cells to PBMC staining tube or 1 drop of negative CompBeads to each bead tube.
(For compensation, positive and negative populations are need in a tube.)

Methods : PBMC staining
- Thaw the PBMC stock 1x10⁷ cells (LN tank 1 rack 4)
- Put cells into 9 mL FACS buffer in 15 mL tube
- Spin down 1,500 rpm x 5 min at room temp.
- Remove sup
- Add 1 mL of FACS Buffer
- Count cells
- Aliquot 50 ul of cell suspension into 1.5 mL tube
- Add 50 ul of Master MiX-1
- Incubate for 30 min at RT covered with foil.
- Add 1mL of FACS buffer.
- Spin down (2,400 rpm x 3 min) and remove sup
- Add 50 ul of unstained cell suspension.
- Spin down (2,400 rpm x 3 min) and remove sup
- Add 500 ul of 1% Fix buffer.

<Staining sample: Rest (100ul of whole blood)>
PCR Room
- Spin down 2,400 rpm x 5 min at room temp.
- Transfer the plasma into screw cap tube. Store at -80C.
- Add 50 ul of FACS buffer.
Note: Now you can go back non-PCR Room
- Add 50 ul of antibody Master Mix 1
- Incubate for 30 min at room temp covered with foil.
- Add 1 mL of 1xRBC lysis buffer and incubate 1 min at room temp.
- Spin down at 2,400 rpm x 3 min and Remove sup
- Add 1 mL of 1xRBC lysis
- Spin down at 2,400 rpm x 3 min and remove sup
- Add 1 mL of FACS buffer.
- Spin down at 2,400 rpm x 3 and remove sup.
- Add 300 ul of Fix buffer.
- Transfer into filtered flow tube (BD Falcon: cat# 352235 ).
- Keep at 4C covered with foil until analysis with flow cytometer.