- Thaw one vial of P#1 organoids and one vial of P#2 organoids, resuspend in 10 ml RMFC media in a 50 ml conical tube, incubate at 37C 10’
- Break up cell clusters with an 18G needle (8 passes)
- Pellet cells, resuspend in PBS + 0.1% BSA, pellet cells
- Resuspend cells in 2 ml 0.05% Trypsin-EDTA, pipet up and down with a p1000 pipet 2’, incubate at 37C 5’, pipet 1’, incubate at 37C 5’, pipet 1’
- Add 8 ml RMFC, add 100 ul 5 mg/ml DNase I, pipet up and down
- Filter through a 20 um mesh
- Count cells
- Pellet and resuspend in at 1 x107 per ml in B2 (PBS + 0.1% BSA + 2 mM EDTA pH 7.4)
- Add each of the following antibodies at 1:50 dilution, except for (d) at 1:200
- CD31 (JC/70A Thermo, IgG)
- CD34 (QBEnd/10 Thermo, IgG)
- CD45 (Bra55 Thermo, IgG)
- Fibroblast Surface Protein (1B10 Sigma IgM)
- Incubate at 4C for 30’ on nutator
- Pool 50 ml of IgM dynabeads and 50 ml of IgG Dynabeads in 1 ml of B2, magnet 1’, remove supernatant (note use 50 µl each for 1 ml or less of cells, scale up accordingly if more)
- Resuspend beads in 100 ml B2, keep on ice Wash cells bound by antibodies 2x with B2, resuspend in 1 ml B2 and add IgG and IgM mixture
- Incubate with occasional mixing at 4C or on ice for 30’
- Add an additional 1 ml of B2, magnet 2’
- Transfer supernatant to a new tube, count total cells
- Pellet cells and resuspend at 1 x 106 per ml in FACS buffer (FB: PBS + 1% CS), divide 200 ml (200K cells) per FACS tube (note put double in unstained sample if possible and double in tube 7). Note that you can get away with less (100K) for samples that aren’t needed to set voltages or compensation.
- Add antibody as indicated below and incubate for 20’ at 4C
- Add 2 ml ice-cold FB to wash, pellet cells, repeat (note for those needing primary, then secondary, you can cut this to one wash to save some time).
- Resuspend all samples in 250 ml FB, keep on ice and in the dark
- At FACS machine add 25 ml of 1:1000 PI (1:1000 dilution of 1 mg/ml) to the indicated samples
General Staining workflow:
All conjugated primaries:
Incubate with antibodies for 20’ at 4C, wash, resuspend
Unconjugated primary only:
Incubate with primary antibody for 20’ at 4C, wash, resuspend in FB
Incubate with secondary antibody for 20’ at 4C, wash, resuspend in 250 µl FB
Unconjugated primary + conjugated primary:
Incubate with primary antibody for 20’ at 4C, wash, resuspend in FB
Incubate with secondary antibody for 20’ at 4C, wash, resuspend in FB
Incubate with conjugated primaries for 20’ at 4C, wash, resuspend in 250 µl FB
Calibur setup
Use unstained sample to set voltages for FSC/SSC and FL1, 2, 3, 4
Use the histogram for each channel and dot plots to set voltages
Set compensation with single stains of each fluorophore
*FITC (FL1) will fluoresce in FL1 and FL2, subtract %FL1 from FL2
*PE (FL2) will fluoresce in FL1, FL2 and FL3, subtract %FL2 from FL1 and FL3
*PI (FL3) will fluoresce in FL2, FL3 and FL4, subtract %FL3 from FL2 (a lot) and FL4 (a little)
*PerCP (FL3) will fluoresce in FL2, FL3 and FL4, subtract %FL3 from FL2 (a little) and FL4 (a lot)
*APC (FL4) will fluoresce in FL3 and FL4, subtract %FL4 from FL3
Collect 30,000+ events for samples to account for dead cells in each tube (25-35%)
CD49f-FITC (Rat IgG2A) BD Cat# 555735 clone GoH3: USE 4 µl
Or CD49f-PE (Rat IgG2A) BD Cat#555736 clone GoH3: USE 4 ml
CD24-PE (mouse IgG2A) BD Cat# 555428 clone ML5: USE 4 µl
Or CD24-FITC (mouse IgG2A) BD Cat#555427 clone ML5: USE 4 µl
EpCAM-APC (mouse IgG1) BD Cat# 347200 clone EBA-1: USE 1 µl
Rat IgG2A-FITC isotype control BD Cat# 553929: USE 4 µl
Mouse IgG2A-PE isotype control BD Cat# 559319: USE 4µl
Mouse IgG1-APC isotype control BD Cat# 340442: USE 1µl
Set up the same samples for each patient (minimally need about 1-1.4 x106 cells)
- 1. Unstained
- 2. Isotype
- 3. FITC-CD24
- 4. PE-CD49f
- 5. PI (25 µl @ before heading to FACS machine)
- 6. APC-EpCAM BD
- 7. FITC-CD24 BD + PE-CD49f + APC-EpCAM (use double cells!)
Post-staining split sample 7 into 2 fractions, A and B, add PI to B
