Egm 2 mv singlequot kit supplements growth factors

HUVEC (Lonza, Breda, The Netherlands) were cultured in EBM-2 medium supplemented with EGM-2 MV Single Quot Kit Supplements & Growth Factors (cat No. CC-3202, Lonza) and used when confluent. Three groups were evaluated: (1) HUVEC pre-incubated for 2 hours with 5 µM atorvastatin (Sigma Aldrich, Saint Louis, USA) and treated with LPS (100 ng/ml) (Sigma Aldrich, Saint Louis, USA), (2) HUVEC treated only with LPS, and (3) unstimulated HUVEC. Supernatants were collected for measuring HMGB1 and interleukin (IL)-8 at baseline, 4 hours and 24 hours. All in vitro experiments were performed twice and in duplicate. Cell viability of HUVEC was checked with 0.2% trypan blue dye (Invitrogen, Carlsbad, USA) on HUVEC treated with 0 µM, 0.1 µM, 1.0 µM, 5.0 µM and 10.0 µM atorvastatin; percentages of living cells were 94%, 95%, 95%, 92% and 91% at 36 hours, respectively. Western blot was used to measure HMGB1 in supernatants as previously described [13] (link) while IL-8 levels were measured by ELISA (R&D Systems, Minneapolis, USA).

Human umbilical vein endothelial cells (HUVEC) (CC2519, Lonza) were cultured in EBM-2 medium supplemented with EGM₂MV SingleQuot Kit Supplements & Growth factors (Lonza) at 37°C and 5% CO₂ by the Endothelial Cell Culture Facility of the UMCG. In all experiments, cells between passage 4-8 were used. HUVEC were seeded and grown overnight to a confluent monolayer.
For the leukocyte adhesion assays, the leukaemia cell line HL-60 (kindly provided by Dr. G. Fey, University of Erlangen, Germany) was cultured in RPMI 1640 medium (Lonza) supplemented with 10% (v/v) FCS.

To mimic the context of sepsis in which inflammation involves the role of endothelial cells and blood cells, we used Primary Human Umbilical Vein Endothelial cells (HUVEC) (Lonza, The Netherlands) as endothelial cells and THP-1 (ATCC, The Netherlands) as monocytes. Pooled donor-HUVEC were purchased from Lonza (C2519A, The Netherlands). Cells were cultured in EBM-2TM medium (Lonza) supplemented with EGM-2 MV SingleQuot Kit Supplements & Growth Factors (Cat. No. 3202, Lonza) and antibiotics 100 IU/ml of penicillin (Astellas Pharma, The Netherlands) and 50 μg/ml of Streptomycin (Rotexmedica GmbH, Germany). Cells were used from passage 3–7 and cultured at 37°C, 5% CO2 and saturating humidity. THP-1 cells (ATCC, The Netherlands) were cultured in Gibco TM RPMI 1640 containing L-glutamine +/HEPES + (Cat. No. 1640 52400-025) supplemented with 10% (v/v) heat-inactivated FBS (Gibco), 1%(v/v) Pen/Strep 10.000 U/ml (Gibco). THP-1 cells were kept at 37°C, 5% CO2 and saturating humidity. Cells were freshly passed twice a week to keep a density of 200.000–800.000 cells/ml and used up to passage 28.