Primary HUVECs tested for contamination were purchased from PromoCell GmbH (Heidelberg, Germany) and cultured as previously described (Hannah et al., 2005 (link)). Human Embryonic Kidney-293 (HEK-293) cells were cultured in Minimal Essential Medium (MEM) Alpha Medium 1x (Invitrogen) supplemented with 10% fetal calf serum (Biosera, Ringmer, UK) and 50 μg/ml gentamycin (Invitrogen) at 37°C, 5% CO2 as previously described (Kiskin et al., 2010 (link)). Secreted VWF propolypeptide (VWFpp) was assayed by specific ELISA as previously described (Hewlett et al., 2011 (link)). Primary antibodies (Abs) along with the dilutions for immunofluorescence or western blotting are given in supplementary Table S1 . All reagents were from Sigma-Aldrich unless otherwise stated. Fura-2/AM was from Invitrogen.
Protocol detail
HUVEC and HEK-293 Cell Culture and VWF Assay
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Alpha minimal essential medium
Antibodies
Cells
Dilutions
Elisa
Embryonic
Fetal calf serum
Fura 2 am
Gentamycin
Human
Immunofluorescence
Kidney
Corresponding Organization :
Other organizations : St George's, University of London, Public Health England, Sanquin, University of Amsterdam
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Variable analysis
independent variables
- Culture conditions for HEK-293 cells (culture medium, serum, and antibiotics)
- Secreted VWF propolypeptide (VWFpp) measured by ELISA
- Primary HUVECs tested for contamination were purchased from PromoCell GmbH and cultured as previously described
- Incubation conditions for HEK-293 cells (37°C, 5% CO2)
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