Flow Cytometry

Dear Juan Zhou,

thank you for your message. I will try to extend the protocol to more details, using our (and internationally valid) standard operation procedure (SOP), but you basically find the most important information in the Material and Methods section of the paper. I am not sure about what you refer to when saying "more detailed protocol for flow cytometry" since this includes different steps and is a rather unspecific request. I have added the SOP for staining cells for flow cytometry below, I hope this helps!

Kind regards,

N. Marquardt

Preparation of PBMC:

1. Transfer blood into 50ml-tube (max 15ml). Mix 1:2 with PBS. Underlay with 1 part Ficoll (1 part blood, 1 part PBS, 1 part Ficoll). 
2. Spin: 800 x g, no break, low acceleration, 20min, Room temperature (RT)
3. Transfer interphase into new 50ml tube
4. Fill up with PBS
5. Spin: 800 x g, full break, full acceleration, 10min, RT
6. Discard supernatant
7. Resuspend cells in FACS Wash (FW) (PBS + 10% FCS + 2mM EDTA), probably transfer to smaller tube (dependent on cell number). 
8. Spin: 200 x g, 10min (7min for 15ml-tube, 5min for 5ml-tube), RT. 
9. Discard supernatant, resuspend cells in 1ml (or more) of FW
10. Count cells

FACS staining:

1. Stain in 96-well V-bottom plate
2. Distribute cells (1-2x10e6 cells/well), pellet the cells (spin 500 x g, 2min, check for cell pellet (!!), discard supernatant after centrifugation)
3. Optional: Add 25ul primary antibody cocktail containing only the mAbs against chemokine receptors, resuspend well, incubate 5-10min in incubator. Proceed then with adding 25ul of the primary cocktail containing mAbs against all other surface markers, so that the total volume is 50ul (= step 4).
4. Add 50ul primary antibody cocktail, 
5. Resuspend (with multichannel pipette if applicable)
6. Incubate 15-20min. Dark in fridge (4°C).
7. Wash the cells: 
   1. Add 120ul FW to each well, resuspend, 
   2. spin 2min 1.800 RPM/500 x g
   3. Check for pellets!
   4. take SN off with multipipette
   5. Resuspend in 180ul FW
   6. Spin, check for pellets, flick out supernatant
8. Add 50ul secondary antibody cocktail
9. Repeat steps 4-6
10. Fix cells: Resuspend in 200ul eBioscience Fix/Perm dilution
11. Incubate for 20min at RT, dark

Spin down. If no intracellular staining (ICS), wash again in FW and finally resuspend in 180ul FW. Transfer to Costar® FACS tubes. Store in fridge.

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