TABLE 2.
Reagents and resources list for MRPEC isolation.
| Antibodies/reagents | Host/clonality | Concentrations | Source | Product ID |
| Part A: Immunofluorescence staining and flow cytometry analysis antibodies/nuclear reagents with specific concentrations used | ||||
| Primary/primary-conjugated antibodies | ||||
| Anti-VE-cadherin antibody (EPR18229)] | Recombinant Rabbit anti-Mouse Monoclonal; accession #P33151 | 1:1,000 | Abcam | ab205336 |
| Anti-PLVAP/PV-1 antibody (MECA-32) | Recombinant Rat anti-PLVAP/PV-1 Monoclonal; accession #Q9BX97 | 1:500 | Abcam | ab27853 |
| Anti-isolectin GS-IB4 (GSL-1) AF488-conjugated antibody | Isolectin GS-IB4, Griffonia simplicifolia, Alexa Fluor 488 Conjugated | 20 μg/ml | Invitrogen | I21411 |
| Anti-CD31/PECAM-1 antibody | Goat anti-Mouse/Rat Polyclonal; accession #Q08481 | 1:100 | Novus | AF3628 |
| Anti-EHD3 Antibody (RR-L) | Mouse anti-Mouse monoclonal IgG1-κ EHD3 antibody (RR-L) | 1:250 | Santa Cruz | sc-100723 |
| Recombinant anti-S100A4 antibody [EPR14639(2)] | Recombinant Rabbit anti-Mouse S100A4 Monoclonal | 1:250 | Abcam | ab197896 |
| Recombinant anti-NG2 antibody (EPR23976-145) | Recombinant Rabbit anti-Mouse NG2 Monoclonal | 1:100 | Abcam | ab275024 |
| Recombinant anti-EpCAM antibody (EPR20532-222) | Recombinant Rabbit anti-Mouse EPCAM Monoclonal | 1:100 | Abcam | ab213500 |
| Secondary antibodies | ||||
| **Donkey anti-Rabbit Alexa Flour 488+ (**used for VE-cadherin IF secondary staining in Figures 4A, B) | Donkey anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Alexa Fluor Plus 488; RRID#AB_2762833 | 1:1,000 | Invitrogen | A32790 |
| **Donkey anti-Goat Alexa Flour 555+ (**used for PECAM1 (CD31) IF secondary staining in Figure 4A) | Donkey anti-Goat IgG (H + L) Highly Cross-Adsorbed Alexa Fluor Plus 555; RRID#AB_2762839 | 1:500 | Invitrogen | A32816 |
| **Donkey anti-Rat Alexa Flour 555+ (**used for PLVAP (PV-1) IF secondary staining in Figure 4B) | Donkey anti-Rat IgG (H + L) Highly Cross-Adsorbed Alexa Fluor Plus 555 | 1:500 | Invitrogen | A48270 |
| **Goat anti-Mouse Alexa Fluor 488 (**used for EHD3 IF secondary staining in Supplementary Figure 2A) | Goat anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 | 1:500 | Invitrogen | A-11001 |
| Nuclear staining | ||||
| Ibidi Mounting Medium with DAPI | – | 300 μl of mounting media/imaging dish | Ibidi | 50011 |
| 4′,6-Diamidino-2-phenylindole, dihydrochloride (DAPI) *FluoroPure grade* | – | Final diluted concentration: 300 nM | Invitrogen | D21490 |
| Flow cytometry antibodies | ||||
| CD146 (LSEC) antibody, PE, REAfinity | Recombinant human IgG1; Human anti-Mouse Monoclonal (REA1064) | 1:50 | Miltenyi Biotec | 130-118-407 |
| CD144 (VE-cadherin) antibody, APC, REAfinity | Recombinant human IgG1; Human anti-Mouse Monoclonal (REA225) | 1:50 | Miltenyi Biotec | 130-102-738 |
| CD309 (VEGFR-2) antibody, APC, REAfinity | Recombinant human IgG1; Human anti-Mouse Monoclonal (REA1116) | 1:50 | Miltenyi Biotec | 130-119-434 |
| CD31 antibody, VioBright B515, REAfinity | Recombinant human IgG1; Human anti-Mouse Monoclonal (REA784) | 1:50 | Miltenyi Biotec | 130-111-544 |
| REA control antibody (S), PE, REAfinity | Recombinant human IgG1; Human anti-Mouse Monoclonal (REA293) | 1:50 | Miltenyi Biotec | 130-113-438 |
| REA control antibody (S), APC, REAfinity | Recombinant human IgG1; Human anti-Mouse Monoclonal (REA293) | 1:50 | Miltenyi Biotec | 130-113-434 |
| REA control antibody (S), VioBright B515, REAfinity | Recombinant human IgG1; Human anti-Mouse Monoclonal (REA293) | 1:50 | Miltenyi Biotec | 130-113-445 |
| CD31 (PECAM-1) monoclonal antibody (390), APC, eBioscience | CD31 (PECAM-1) Rat IgG2a kappa; Rat anti-Mouse Monoclonal Antibody (390) | 1:50 | eBioscience | 17-0311-82 |
| Rat IgG2a kappa isotype control (eBR2a), APC, eBioscience™ | Rat IgG2a kappa Isotype Control; Rat anti-Mouse Monoclonal (eBR2a) | 1:50 | eBioscience | 17-4321-81 |
| Part B: All other materials/reagents | ||||
| Digitonin | Used for cell permeabilization (PLVAP and GSL-1 IF-staining only) at a final concentration of 10 μg/ml (w/v) in 1× DPBS+/+ | 10 μg/ml (w/v) | Abcam | ab141501 |
| 25G ×0.75 in. BD Vacutainer® Safety-Lok | Remove blood collection tube/needle and instead attach the 25G needle w/tubing to a 50 ml syringe | – | BD | BD-367285 |
| BD Luer-Lok Syringe sterile, single use, 50 ml | – | – | BD | BD-309653 |
| TC20 Automated Cell Counter | – | – | Bio-Rad | 1450102 |
| 5 ml Round Bottom Polystyrene Test Tube, with Cell Strainer Snap Cap | – | – | Corning | 352235 |
| Disposable Sterile Bottle-Top Filters with 0.22 μm Membrane 45 mm | – | – | Corning | 431118 |
| Disposable Sterile Bottle-Top Filters with 0.22 μm Membrane 33 mm | – | – | Corning | 431117 |
| Primaria 35 mm Cell Culture Dishes | – | – | Corning | 353801 |
| Matrigel basement membrane matrix, growth factor reduced, LDEV-free | – | – | Corning | 354230 |
| Polystyrene Serological Pipets, 5 ml | Sterile, single wrapped RNase-/DNase-free and BSE/TSE-free | – | Corning | 356543 |
| Polystyrene Serological Pipets, 10 ml | Sterile, single wrapped RNase-/DNase-free and BSE/TSE-free | – | Corning | 356551 |
| Polystyrene Serological Pipets, 25 ml | Sterile, single wrapped RNase-/DNase-free and BSE/TSE-free | – | Corning | 356525 |
| Cytiva Percoll™ PLUS Centrifugation Media | – | – | Cytiva | 17544502 |
| Easy Reader 15 ml Conical Polypropylene Centrifuge Tubes | – | – | Fisherbrand | 05-539-4 |
| Easy Reader 50 ml Conical Polypropylene Centrifuge Tubes | – | – | Fisherbrand | 05-539-13 |
| Sterile Cell Strainer 40 μm | – | – | Fisherbrand | 22-363-547 |
| HBSS, calcium, magnesium, no phenol red | – | – | Gibco | 14025 |
| DPBS, calcium, magnesium, glucose, pyruvate | – | – | Gibco | 14287072 |
| Penicillin-Streptomycin (10,000 U/ml) | – | 1% of final volume when indicated | Gibco | 15140-122 |
| Polypropylene Graduated Microcentrifuge Tube with Snap Cap, 1.5 ml, Natural | – | – | Globe Scientific | 111558 |
| Advanced TC Treated Sterile Cell Culture Multi-Well Plates | – | – | Greiner Bio-One | 662960 |
| μ-Dish 35 mm, high, ibiTreat – Tissue Culture Treated Polymer Coverslip | – | – | Ibidi | 81156 |
| Image-iT Fixative Solution (4% formaldehyde, methanol-free) | Used in immunofluorescence experiments for cell fixation step | – | Invitrogen | R37814 |
| BlockAid Blocking Solution | Used for cell blocking steps during immunofluorescence experiments | – | Invitrogen | B10710 |
| Image-iT FX Signal Enhancer ReadyProbes™ Reagent | Used for cell blocking steps during immunofluorescence experiments | – | Invitrogen | R37107 |
| LS Magnetic Columns | – | – | Miltenyi | 130-042-401 |
| CD146 (LSEC) MicroBeads mouse | Mixed end-over-end on microcentrifuge rotator in 4c | 1:35 | Miltenyi | 130-092-007 |
| CD326 (EPCAM) MicroBeads, mouse | Mixed end-over-end on microcentrifuge rotator in 4c | 1:30 | Miltenyi | 130-105-958 |
| MACS SmartStrainers (70 μm) | Used for cell straining prior to accutase digestion steps | – | Miltenyi | 130-098-462 |
| Pre-Separation Filters (70 μm) | Used with LS columns and MACS purification steps | – | Miltenyi | 130-095-823 |
| FcR Blocking Reagent, mouse | Used to block FcR on cells to reduce possibility of non-specific binding of magnetic microbeads | 1:50 | Miltenyi | 130-092-575 |
| gentleMACS Dissociator | Used for tissue digestion steps | – | Miltenyi | 130-093-235 |
| gentleMACS C Tubes | – | – | Miltenyi | 130-093-237 |
| Fibronectin Solution | Human Plasma Fibronectin | 10 μg/ml/dish | PromoCell | C-43060 |
| Fetal bovine serum | – | See text for more details (5–10% v/v) | Lonza | CC-3202 |
| Triton X-100 | Used for cell permeabilization at a final concentration of 0.1% (v/v) in 1× DPBS+/+ | 0.1% (v/v) | Sigma | T8787-100ML |
| Millex GP syringe filter unit, 0.22 μm, sterile | – | – | Sigma-Aldrich | SLGP033RS |
| Accutase® solution | Used for second round of enzymatic digestion and removing cells from culture dishes | – | Sigma-Aldrich | A6964-100ML |
| Samco General Purpose Transfer Pipettes, Standard Bulb, 7.7 ml, Ind. Pack. | – | – | Thermo Fisher | 202-1SPK |
| TIPONE FILTER TIPS (1000 XL Graduated 10 Rack of 96) | – | – | USA Scientific | 1122-1830 |
| TIPONE FILTER TIPS (200 Graduated 10 Rack of 96) | – | – | USA Scientific | 1122-8810 |
| TIPONE FILTER TIPS (10 XL Graduated 10 Rack of 96) | – | – | USA Scientific | 1122-1830 |
FluoroPure grade means ≥98% pure by manufacturer’s HPLC analysis.