Flow‐activated cell sorting (FACS)‐sorted CD27− and CD27+ memory CD19+ B cells from buffy coat–derived PBMCs were placed on 96 permeable transwell plates with a 0.3μm pore size (2 × 105 cells/well; Corning, Amsterdam, the Netherlands). B‐cell migration toward medium or CXC chemokine ligand (CXCL)10 (900ng/ml; R&D Systems, Abingdon, UK) was analyzed after 3 hours at 37°C. In addition, 2.5–5 × 105 memory B cells were placed on confluent monolayers of human brain endothelial cells (hCMEC/D3) on 5μm pore size transwell plates (Corning) coated with collagen, and migration was analyzed after 5 hours.22 Percentages of memory B‐cell subsets were compared before and after transmigration using flow cytometry.
Hcmec d3
Manufactured by Corning
Sourced in United States
HCMEC/D3 is a human cerebral microvascular endothelial cell line derived from brain tissue. It is designed for use in in vitro studies related to the blood-brain barrier.
Automatically generated - may contain errors
Lab products found in correlation
Cxcl10, by R&D Systems (1 mentions)
T75 culture flask, by Corning (1 mentions)
Hdmec, by PromoCell (1 mentions)
Human umbilical vein endothelial cells (huvec), by Corning (1 mentions)
Hcmec d3, by Cedarlane (1 mentions)
Huvecs, by ScienCell (1 mentions)
Hpc pl, by PromoCell (1 mentions)
Hcmec d3, by Merck Group (1 mentions)
Pericyte growth medium 2, by PromoCell (1 mentions)
Endogro medium, by Merck Group (1 mentions)
Human tnf α, by Abcam (1 mentions)
3 protocols using hcmec d3
1
Memory B Cell Migration Assay
2
Culturing Endothelial Cell Lines
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HUVECs (ScienCell, USA), hCMEC/D3 (Cedarlane, Canada), hBMEC (Cell Systems, USA), and hDMEC (Promocell, Germany) were cultured in endothelial cell medium (ScienCell, USA) containing 10% fetal bovine serum (FBS), 1% penicillin‐streptomycin (P/S), and 1% endothelial cell growth supplement (ECGS). Frozen stocks were prepared for all cell types after expanding cells in vendor‐recommended media. Frozen stocks of the following passages were thawed and cultured in T75 culture flasks (Corning, USA), where Passage 1 for all cell type indicates the initial stock received from the vendor; HUVEC: Passage 4, hCMEC/D3: Passage 5–7, hBMEC: Passage 6, hDMEC: Passage 3, 2–3 d prior to cell seeding. All cells were cultured in a humidified 37 °C incubator with 5% CO2.
3
In Vitro Blood-Brain Barrier Model
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Human cerebral microvascular endothelial cells D3 (hCMEC/D3, Merck Millipore, Germany) were cultivated as advised by the manufacturer in EndoGRO™ medium (Merck Millipore, Germany) with 1 ng/mL FGF-2. Human pericytes (hPC-PL, PromoCell, Germany) were cultured according to the manufacturer´s instructions in Pericyte Growth Medium 2 (Promocell, Germany). For BBB models, 25,000 hCMEC/D3 cells were seeded on the lower side of a 6 mm polyester membrane cell culture insert with 0.4 μm pores (Corning® Transwell®, USA) and left to incubate and attach for 1 h. After placing the inserts into the well, 50,000 hPC-PL were seeded into the upper compartment. This generated a blood compartment on the endothelial side and a CNS compartment facing the pericytes (Fig. 1A). The model was left to establish for 15 days at 37°C with 5% CO 2 . Further experiments were conducted if stable and consistent resistance measurements indicated an intact barrier. To induce inflammation, the models were incubated with human TNFα (abcam, Great Britain) at a concentration of 20 ng/ml in medium for 4 h under standardized cell culture conditions [20] .
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