The HMEC-1 cell line was cultured as previously described and used between passages 8 and 18 (26 (link)). Coculture experiments were carried out with ECs and non-HLA matched PBMCs, as reported (28 (link)). PBMCs were isolated from healthy donor blood samples (obtained in accordance with institutional regulations from the Etablissement Français du Sang, Paris, France) by Ficoll density gradient separation (Eurobio, Les Ulis, France).
Ficoll density gradient separation
Manufactured by Eurobio Scientific
Sourced in France
Ficoll density gradient separation is a laboratory technique used for the isolation and purification of cells, proteins, and other biological molecules. It utilizes a Ficoll polymer solution to create a density gradient, which allows for the separation of different components based on their density. This method is commonly used in various applications, such as isolating specific cell types from complex biological samples.
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3 protocols using ficoll density gradient separation
1
HMEC-1 Coculture with PBMCs
2
Culturing Human Dermal Microvascular and Brain Endothelial Cells
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The human dermal microvascular endothelial HMEC‐1 cell line, provided by A. Kesikli (University of Regensburg, Germany) was cultured in complete MCDB 131 medium, composed of MCDB 131 medium (Thermofisher Scientific) supplemented with 12.5% fetal bovine serum (FBS), hydrocortisone 1 μg/ml (Sigma‐Aldrich), epidermal growth factor (EGF) 10 ng/ml (BD Biosciences), 6 mM Glutamine and used between passages 12 and 20.19 , 20 The hCMEC/D3 cell line was cultured in complete EndoGRO Basal Medium (Merck), composed of EndoGRO Basal Medium supplemented with 0.2% EndoGRO LS Supplement, recombinant EGF 5 ng/ml, hydrocortisone hemisuccinate 1 μg/ml, heparin sulfate 0.75 U/ml, ascorbic acid 50 μg/ml, l ‐Glutamine 10 mM, 5% FBS and used between passage 30 and 40.26 Coculture experiments were carried out with ECs and peripheral blood mononuclear cells (PBMCs), as reported elsewhere.19 , 20 Briefly, PBMCs were isolated from healthy donor blood samples (obtained in accordance with institutional regulations from the Etablissement Français du Sang) by Ficoll density gradient separation (Eurobio). PBMCs were maintained in complete RPMI 1640 medium (Thermofisher Scientific), composed of RPMI 1640 with 10% human AB serum and a final concentration of HEPES 10 mM, sodium pyruvate 1× (Eurobio), glutamine 2 mM.
3
Immune response to Aspergillus hyphae
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Conidia (1x104/well) were incubated in 96-well microtiter plates with 30 μg/mL rfhSP-D or FL-SP-D for 16 h at 37 °C. Following, the culture medium was removed, hyphae were washed with PBS, and fixed in 2.5% PFA overnight at 4 °C. Control hyphae were prepared similarly in the absence of SP-D. Blood samples of healthy donors were obtained from the Etablissement Français du Sang (Paris, France) with written informed consent as per the guidelines provided by the Institutional Ethics Committee, Institut Pasteur (convention 12/EFS/023). Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples by Ficoll density-gradient separation (Eurobio, France). PBMCs (2x106 cells/well) in complete RPMI [containing 10% pooled normal human serum (ZenBio)] were added to the fixed hyphae and incubated at 37 °C, 5% CO2. The cell-free supernatant was collected after one and five days of co-incubation and stored at −20 °C. TNF-α, IL-1β, IL-6 (pro-inflammatory), IL-10 (an anti-inflammatory cytokine) and IL-8 (chemokine) levels in the culture supernatants were measured by using ELISA kits (DuoSet, R&D Systems).
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