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5 protocols using mesenchymal stem cell growth supplement

1

Culturing Human Hair Follicle Cells

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Human hair follicle dermal papilla cells (hFDPCs) were purchased from PromoCell
(Heidelberg, Germany). The hFDPCs were cultured in FDPC growth medium
supplemented with 4% (v/v) fetal calf serum, 0.4% (v/v) bovine pituitary
extract, human recombinant basic fibroblast growth factor (1 ng/mL), and human
recombinant insulin (5 μg/mL; PromoCell). Human hair germinal matrix
cells (hGMCs) were obtained from ScienCell Research Laboratories (Carlsbad, CA,
USA). The hGMCs were cultured in mesenchymal stem cell medium supplemented with
5% (v/v) fetal bovine serum, and 1% (v/v) mesenchymal stem cell growth
supplement (ScienCell Research Laboratories). Both hFDPCs and hGMCs were
cultured at 37°C in a humidified atmosphere of 5 % CO2.
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2

Adrenal Cortical Cell Culture Infection

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Primary human adrenal cortical cells (HAdCCs; ScienCell, Carlsbad, CA, USA) were used; cell type was confirmed by an immunofluorescence antibody assay showing the presence of glial fibrillary acidic protein (GFAP; present in adrenal cells in the cortex) and absence of tyrosine hydroxylase (TH; present in adrenal chromaffin cells in the medulla). HAdCCs were seeded at 5000 cells/cm2 in mesenchymal medium containing 5% fetal bovine serum (FBS), 1% mesenchymal stem cell growth supplement, and 1% 100X penicillin-streptomycin (ScienCell). After 24 h, media was changed to media with 0.1% FBS and 1% 100X penicillin-streptomycin and was replenished every 72 h for 7 days, establishing quiescence (qHAdCC). On day 7, uninfected (mock) or VZV-infected qHAdCCs (60 plaque-forming units/cm2; VZV Gilden strain [GenBank accession number MH379685] [7 (link)]) were added to qHAdCCs. For analysis, cells were harvested at 1, 2, or 3 days post-infection (DPI), and conditioned supernatants were collected at 3 DPI.
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3

Isolation and Culture of Pancreatic CAFs and MSCs

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Pancreatic CAFs were isolated from primary tumour tissues by digesting minced tissue with 0.4 U/mL dispase and 120 U/mL collagenase (ThermoFisher Scientific, Paisley, UK) at 37 °C and culturing in PCA medium (DMEM supplemented with 7.2 μg/mL insulin (Sigma-Aldrich), 1 μg/mL hydrocortisone (Sigma-Aldrich), 100 U/mL penicillin, 100 U/mL streptomycin, 0.25 μg/mL amphotericin B, 2 mM L-glutamine, and 20% FBS). Human MSCs were purchased from ScienCell (Carlsbad, CA, USA) and cultured in mesenchymal stem cell media (MSCM, ScienCell) which consists of basal medium 5% FBS and Mesenchymal Stem Cell Growth Supplement (ScienCell).
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Isolation and Characterization of Mouse Bone Marrow-Derived Mesenchymal Stem Cells and Neutrophils

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Mouse bone marrow-derived MSCs were generally isolated from the tibia and femoral marrow compartments and were identified by flow cytometry as described previously.18 (link) The cells cultured in MSC culture medium (Science Cell, 7501) with 1% mesenchymal stem cell growth supplement (Science Cell,7552), 5% fetal bovine serum (Science Cell,0025) and 1% Penicillin/Streptomycin (Science Cell,0503). The cell incubator was maintained at 37°C with 5% CO2 and 95% humidity. Every 2–3 days the culture medium was changed and Trypsin-EDTA (0.25%) (Gibco, 25200056) was used for passaging.
Neutrophils were isolated from bone marrow of 4-week-old C57BL/6J mice. The femur and tibia were resected intactly and cut the two ends of the bone in a clean bench to flush the bone marrow out. After treatment with red cell lysis buffer, the neutrophils were isolated using Mouse neutrophil isolation kit (Miltenyi Biotec, 130–097-658) according to the manual. The isolated neutrophils were cultured in RPMI1640 medium supplemented with 10%FBS for future use.
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5

Preparation of Bone Marrow-Derived Mesenchymal Stem Cell Conditioned Media

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BM-MSCs (7500, ScienCell, USA) were cultured in Mesenchymal Stem Cell Medium (7501, ScienCell, USA) composed of basal growth medium, 5% FBS (0025, ScienCell, USA), 1% Mesenchymal Stem Cell Growth Supplement (7552, ScienCell, USA), and 1% P/S (0503, ScienCell, USA). The cell culture media was changed every 3 days. BM-MSCs at passage 9 were seeded in the culture dish for 16 h and washed three times with PBS. BM-MSCs were cultured in basal medium for an additional 24 h at 37 °C in a 5% CO2 atmosphere. The medium was collected and concentrated 40x using a 10 kDa centrifugal filter (Amicon Ultra-15, Millipore, USA) [25 ]. The CM from human BM-MSCs (BM-MSC CM) was stored at 4 °C until use.
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