Supplementary Information for HBMEC Isolation

Before you begin, you will need to prepare?why;

Solutions:

1.       DMEM-S: DMEM containing 2% FCS

2.        10x PBS/phenol red

3.       30% Dextran in dH₂O, autoclave, add 10xPBS with phenol red (eg. 22.5ml 30% dextran + 2.5 ml 10xPBS/phenol red (sometimes, dextran is a bit acidic, hence the phenol red; if it turns yellow add 0.1M NaOH to neutralise)

4.       A solution of 1mg/ml collagenase/dispase in DMEM-S, and 0.1mg/ml DNaseI (collagenase/dispase soln; Boehringer cat# 269 638)

5.       Basal RPMI 1640: nothing added

6.        Culture medium: RPMI 1640 containing the following:

       10% fetal bovine serum (FBS), 10% NuSerum, endothelial cell growth supplement (ECGS; 30¦Ìg/ml) (Collaborative Biomedical Products, B&D, Bedford, MA), heparin (5U/ml), L-glutamine (2mM), sodium pyruvate (1mM), MEM non-essential amino acids, MEM vitamins, penicillin and streptomycin (100 U/ml) (Irvine Scientific, Irvine, CA).

        Make up 500ml RPMI with everything except heparin & ECGS, these should be added to a small volume of medium, about 50-100ml, as the reagents are very expensive. Note that L-Glutamine degrades and should also be added relatively freshly.

Autoclaved:

1.      Dounce homogenizer with tight fitting

2.      Glass beads (212-300microns; Sigma #G9143) washed with dH2O before autoclaving

      Glass bead column: made from a 10ml syringe: Cut bottom off, tape fine mesh around it and fill with the glass beads (about 1ml depth). Autoclave.