TABLE 1.
Compositions and components of buffers and solutions for primary MRPEC isolation.
| Materials/reagents | Notes | Concentrations | Source | Product ID |
| Part A: Formulation of collagenase type I enzymatic tissue digestion buffer* | ||||
| DMEM/F-12, GlutaMAX supplement, Media | – | Used as supplied by manufacturer | Gibco | 10565018 |
| Collagenase, Type I, powder | As manufacturer lot numbers vary in enzymatic activity, adjust quantity to enzymatic activity units | ∼1.25 mg/ml (∼350 enzymatic activity units) | Gibco | 17100-017 |
| DNAse-1 | DNase I recombinant, RNase-free | 100 U/ml | Roche | 4716728001 |
| Bovine serum albumin (BSA) | Use BSA heat shock fraction-V, protease free, fatty acid free, essentially globulin free, pH 7, ≥98% | 1 mg/ml | Sigma-Aldrich | A7030-50G |
| Part B: Formulation of Percoll-Plus solution* | ||||
| DMEM/F-12, HEPES, Media | – | 12.5/25 ml (v/v) | Gibco | 11-330-057 |
| Percoll PLUS Centrifugation Media | Used for gradient centrifugation steps | 11.25/25 ml (45% v/v) | Cytiva | 17544502 |
| 20× phosphate buffered saline, powder, pH 7.4 | Prepare a 20× PBS stock solution by adding one packet of PBS powder to 50 ml of filtered ultrapure water | 562.5 μl/25 ml (v/v) | Sigma-Aldrich | P3813-5X10PAK |
| Ultra-Pure DNase/RNase-Free Distilled Water | – | 687.5 μl/25 ml (v/v) | Invitrogen | 10977023 |
| Part C: Formulation of magnetic microbead solution* | ||||
| 1× phosphate buffer saline (PBS), pH 7.4 | Without Ca2+ and Mg2+ (PBS–/–) | 400 ml | Gibco | 10010049 |
| Ethylenediaminetetraacetic acid (EDTA) | A 500 mM stock solution was initially prepared and then diluted to a final concentration of 2 mM | 2 mM [1.6 ml (500 mM stock) / 400 ml] | Sigma-Aldrich | E9884-1Kg |
| Bovine serum albumin (BSA) | Use BSA heat shock fraction, protease free, fatty acid free, essentially globulin free, pH 7, ≥98% | 2 g (0.5% w/v) | Sigma-Aldrich | A7030-50G |
| Part D: Formulation of MV-EC specific cell culture media* | ||||
| EGM™-2MV (Microvasculature Endothelial Cell Growth Medium-2) BulletKit™ | Product contains: EBM™-2 Basal Endothelial Cell Medium and EGM™-2MV SingleQuots™ Supplement Pack | Used/prepared as supplied by manufacturer | Lonza | CC-3202 |
| Heparin sodium solution | Heparin sodium USP at 1,000 U/ml | 0.75 U/ml (375 μl/500 ml of media) | Mylan | NDIC: 67457-385-99 |
| Mouse VEGF-164 Recombinant Protein | Lyophilized from a 0.2 μm filtered solution in PBS | 50 ng/ml (25 μg/500 ml of media) | R&D Systems | 493MV025CF |
| Part E: Formulation of FACS buffer* | ||||
| 1× phosphate buffer saline, pH 7.4 | Without Ca2+ and Mg2+ (PBS–/–) | 400 ml | Gibco | 10010049 |
| Ethylenediaminetetraacetic acid (EDTA) | A 500 mM stock solution was initially prepared and then diluted to a final concentration of 2 mM | 2 mM (1.6 ml of 500 mM stock/400 ml) | Sigma-Aldrich | E9884-1Kg |
| 100% fetal calf serum | – | 4 /400 ml (1% v/v) | Lonza | CC-3202 |
*As the volume of each buffer required for this protocol will vary depending on the number of mice being utilized, scale-up/down the final volume and concentrations proportionally based on this table.