Ebm 2 microvascular endothelial cell growth medium 2 singlequots supplements and growth factors

Primary human lung microvascular cells (Applied Cell Biology Research Institute, ACBRI 468) were seeded in EBM-2 Microvascular Endothelial Cell Growth Medium-2 SingleQuots Supplements and Growth Factors (Lonza) on a collagen-coated 8-well chamber slide (Watson). Various combinations of Fbg, CitFbg, and SAA1 (50 ng/mL, 200 μL) were added to the culture medium twice in a 7 day-culture. The conditioned media were centrifuged at 20,000×g and stained with anti-citrulline (0.5 mg/mL, bs-1053R) and anti-SAA1 antibodies (0.5 mg/mL, ab207445) followed by Alexa Flour 488-conjugated secondary antibody (1:400, A21206, Thermo). Protein aggregations were gently washed, and all aggregations were dried up on the slide to be further analyzed under a confocal microscope (Leica TCS SP8, Heidelberg, Germany).

Primary human lung microvascular cells (Applied Cell Biology Research Institute, ACBRI 468) were cultured in EBM™-2 Microvascular Endothelial Cell Growth Medium-2 SingleQuots™ Supplements and Growth Factors (Lonza). After becoming subconfluent in a 10 cm dish, human TCMs were added at 15–20% and incubated for 24 h. The culture medium was then gathered as lung EC-CMs. Primary hPBMCs (Lonza CC-2702) were cultured in LGM-3 medium (Lonza) with 200 IU/mL IL2. Cells were received from full consent donors with IRB-approved protocols.