Can I use the Growth Medium instead of TNS to stop the trypsin when splitting the cells?

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.

Which medium do PromoCell recommend to use on HUVEC - Endothelial Cell Growth Medium or Endothelial Cell Growth Medium 2?

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.

What is the exact localization of Human Dermal Lymphatic Endothelial Cells (HDLEC)? What medium should I use to culture them?

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).

I am planning to use Endothelial Cell Growth Medium (C-22010) to grow my HUVEC. Do I have to supplement the medium with additional factors like e.g. FCS?

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.

What's the difference between PromoCell HUVEC single donor (C-12200) and HUVEC-pooled (C-12203)?

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).

What is the source of the heparin that comes with the Endothelial Cell Growth Media (C-22010/C-22011/C-22020)?

The source of our heparin is ex porcine mucosa.

How often should I change the growth medium when culturing PromoCell Normal Human Cells?

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.

How long is it before the osteoblasts produce mineralized nodules?

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.

Is it possible to mineralize PromoCell osteoblasts?

Yes, it is possible. Short protocol: Plate human osteoblasts in Osteoblast Mineralization Medium on collagen I coated TC vessels. Incubate the cells for 17-21 days and change the medium every third day. Be careful not to disturb the cell monolayer. Fix the cells. The calcium deposition can be visualized by von Kossa or Alizarin Red staining. More detailed information on osteoblast mineralization and Alizarin Red S staining can be found in the attached Application Note.

We are culturing human coronary artery smooth muscle cells (HCASMC) from your company. Are these cells obtained from large arteries like LAD or left Circumflex or from small branches of LAD etc. ?

Our HCASMC (C-12221) are isolated from the large arteries, i.e. from Right coronary artery Left main coronary artery Circumflex coronary artery and Left anterior descending coronary artery.

What's the difference between trypsin and accutase?

Both, trypsin and accutase represent mixtures of different proteolytic enzymes. Trypsin is prepared from porcine pancreas, accutase from invertebrates. Accutase can replace trypsin for the detachment and dissociation of anchorage-dependent cells from surfaces and can also be used on suspension cells to reduce clumping in preparation for counting. The advantages of accutase over the traditional trypsin treatment are that it is more gentle and less damaging to cells (leading to increased viability) and does not contain any mammalian or bacterially derived proteins. Accutase is more thermolabile than trypsin and usually doesn't require an inactivation step.

Technical library - RESOURCES

General protocol for recovery of anchorage-dependent primary cells: Remove vial from liquid nitrogen, transport it on dry ice to the cell culture lab. Thaw in a 37°C waterbath for approx. 2 min, until it is just defrosted. Keep the vial immersed in water until just below the screw cap during thawing and only remove it shortly after approx. 90 sec to check the progress. Carefully disinfect the vial with plenty of 70% EtOH under the laminar flow hood and aseptically transfer the thawed suspension into an appropriate TC dish with growth medium (pre-warmed in the incubator for > 30 min). The cells usually attach within a few hours. Perform media change after 24 hrs at the latest to remove residual DMSO from the freezing media. Additional information can be found in the instruction manuals of our cells.