Technical library

• FBS (or BSA) can be substituted for HSA, however we do not recommend this as the FBS will lead to unpreferable immunologic stimulation of human macrophages.

• The addition of EDTA to the PBS is to further augment the “anti-clumping” activity of the Ca²⁺/Mg²⁺ free PBS.

All our Human Uterine Fibroblasts (HUF) are prepared from the uterine wall.

A concentration > 10% FBS is needed to completely inactivate the trypsin. As most of PromoCell's growth media are serum-reduced or serum-free, the use of a trypsin inhibitor like TNS is highly recommended.

The standard medium for isolation and propagation of our HUVEC, HUAEC, HPAEC, and HSaVEC is Endothelial Cell Growth Medium (C-22010). It contains ECGS, an extract from bovine hypothalamus which has mitogenic effects on endothelial cell proliferation. Scientists who prefer a more defined Growth Medium can use Endothelial Cell Growth Medium 2 (C-22011). In this medium, ECGS is replaced by VEGF, IGF, and additional bFGF and EGF to stimulate endothelial cell growth.

PromoCell's HDLEC are isolated from skin (dermis). When isolated from juvenile donors (C-12216), the exact localization is foreskin. When derived from adult donors (C-12217), the localization depends on the type of surgery, e.g. breast or temple. You can find the information on the exact localization in the Certificate of Analysis.

Our HDLEC are tested to be positive for CD31, podoplanin, and ac-LDL uptake and are delivered in P2. The recommended culture medium is Endothelial Cell Growth Medium MV2 (C-22022).

PromoCell's Endothelial Cell Growth Medium (C-22010) is a complete medium that can be used for the culture of HUVEC after addition of the SupplementMix. Addition of extra FCS is not necessary. The SupplementMix contains FCS (2% v/v final concentration), recombinant growth factors, hormones, and a bovine brain extract that together have mitogenic effects on endothelial cells.

Our HUVEC single donor (C-12200) are isolated from a single umbilical cord, propagated in primary culture, and frozen down at subconfluency. For the preparation of HUVEC-pooled (C12203), we simultaneously isolate the cells from 2-4 umbilical cords and grow them in separate tissue culture dishes. The cells are pooled after trypsinization given that their growth rates are comparable. After thawing, our HUVECs (single donor and pooled) are both in P1. The recommended media are Endothelial Cell Growth Medium (C-22010) or Endothelial Cell Growth Medium 2 (C-22011). With respect to cell growth, HUVEC-pooled tend to have a more heterogeneous morphology with slightly more elongated cells but the doubling times are comparabel for both types (typically 18-36 hrs per doubling).

The source of our heparin is ex porcine mucosa.

Generally, the medium should be changed every 2-3 days. Please note: Following thawing, the first medium change should be performed after 16-24 hours to prevent cell damage due to residual freezing medium.

For mineralization assays, HOB are cultured in Osteoblast Mineralization Medium (C-27020). Mineralization can be detected after approximately 3 weeks by incorporation of Ca-45, or it can be visualized by von Kossa or Alizarin Red staining for calcium.