LAD-2 cells (National Institute of Allergy and Infectious Diseases, Bethesda, MD, USA) were maintained in StemPro-34 medium (Life Technologies, Grand Island, NY, USA) supplemented with 2 mM l -glutamine, 100 U/mL penicillin, 50 μg/mL streptomycin, and 100 ng/mL recombinant human stem cell factor (R&D Systems). LAD-2 cells were sensitized using 100 ng/mL biotinylated-IgE (BioPorto Diagnostics, Hellerup, Denmark) with or without 5 µg/mL EETs. Cells were stimulated with 100 ng/mL streptavidin peroxidase (Sigma-Aldrich) in Tyrode’s buffer containing 0.1% BSA. Total β-hexosaminidase was obtained via lysing of LAD-2 cells in 0.1% Triton X-100 in PBS. The supernatants were collected and incubated with an equal volume of 4 mM p-nitrophenyl N-acetyl-β-d -glucosamide (Sigma-Aldrich) in citrate buffer for 1 h. The reactions were stopped via the addition of 0.4 M glycine buffer, and signals were read at a wavelength of 405 nm. Levels of TNF-α and MCP-1 (R&D Systems) were measured using ELISA kits according to the manufacturer’s protocol.
Stempro 34 medium
Manufactured by Thermo Fisher Scientific
Sourced in United States
StemPro™-34 medium is a serum-free, defined, and specialized medium designed for the culture and expansion of human hematopoietic stem and progenitor cells.
Automatically generated - may contain errors
Lab products found in correlation
Interleukin 3 (il 3), by Thermo Fisher Scientific (14 mentions)
L glutamine, by Thermo Fisher Scientific (12 mentions)
Stem cell factor (scf), by Thermo Fisher Scientific (12 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (11 mentions)
Percoll density gradient medium, by GE Healthcare (8 mentions)
Cytotune ipsc 2.0 sendai reprogramming kit, by Thermo Fisher Scientific (8 mentions)
Stemmacs ips brew xf medium, by Miltenyi Biotec (8 mentions)
Penicillin, by Thermo Fisher Scientific (7 mentions)
Streptomycin, by Thermo Fisher Scientific (6 mentions)
Stempro 34 sfm medium, by Thermo Fisher Scientific (6 mentions)
Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (4 mentions)
Iwr 1, by Merck Group (4 mentions)
Stem cell factor (scf), by Novoprotein (4 mentions)
Recombinant human stem cell factor, by R&D Systems (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Iwp 2, by Merck Group (3 mentions)
Basic fibroblast growth factor (bfgf), by R&D Systems (3 mentions)
Ascorbic acid, by Fujifilm (3 mentions)
Basic fibroblast growth factor (bfgf), by ReproCELL (3 mentions)
Ascorbic acid, by Merck Group (3 mentions)
56 protocols using stempro 34 medium
1
Mast Cell Degranulation Assay
2
Cultivation of Human Mast Cell and HEK293 Lines
3
Erythroblast Expansion and Differentiation
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HEK-293T and NIH3T3 cells were cultured in DMEM (Invitrogen, Breda, The Netherlands) supplemented with 10% FCS, 100 IU/ml penicillin and 100 µg/ml streptomycin. Mouse liver was isolated from a C57/B6 mouse 8 weeks of age. The I/11 erythroblast line was cultured in StemPro-34 medium (Life Technologies) as described previously [55] (link). For expansion, the medium was supplemented with 0.5 U/ml Epo (Ortho-Biotech, Tilburg, The Netherlands), 100 ng/ml SCF (supernatant of CHO producer cells), and 10−6 M dexamethasone (Dex; Sigma-Aldrich). Differentiation was induced in medium containing 2 U/ml Epo, 0.5 mg/ml holo-transferrin. Cell numbers and cell size distribution were determined using an electronic cell counter (CASY-1; Roche). Cell morphology was analyzed in cytospines stained with histological dyes and neutral benzidine [28] (link), using an OlympusBX40 microscope and OlympusDp50 CCD camera, and Viewfinder Lite 1.0 acquisition software.
4
Generation of Human Mast Cells from Cord Blood
5
Culturing HEK293T, LAD2, and LUVA Cells
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HEK293T cells were cultured in DMEM medium supplemented with 2 mmol/L glutamine, 10% fetal bovine serum and Penicillin/Streptomycin (50 units/ml) (all Life Technologies) in a humidified 5% CO2 37 oC incubator. LAD2 cells (a kind gift from Dr. Arnold Kirshenbaum, NIAID, NIH, USA26 (link)) and LUVA cells (a kind gift from Dr. John Steinke, University of Virginia, USA27 ) were cultured in StemPro-34 medium supplemented with with L-glutamine (2 mmol/L), Pen/Strep (50 IU/ml) and with or without stem cell factor (SCF) (100 ng/ml) (all Life Technologies), respectively. Cells were hemidepleted weekly with fresh medium.
6
Mast Cell Degranulation Assay with UCA
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Laboratory of Allergic Disease 2 (LAD-2) mast cells were kindly provided by Dr. Arnold Kirshenbaum (National Institute of Allergy and Infectious Diseases, Bethesda, MD, USA). Cells were maintained in StemPro-34 medium (Life Technologies, Grand Island, NY, USA) supplemented with 2 mM L-glutamine, 100 U/mL penicillin, 50 μg/mL streptomycin, and 100 ng/mL recombinant human stem cell factor (R&D Systems, Minneapolis, MN, USA) as described previously43 (link).
LAD-2 cell degranulation was evaluated by the β-hexosaminidase release test44 . Cells were treated for 16 h with cis- or trans-UCA with or without simultaneous sensitisation with biotinylated-IgE (100 ng/mL, BioPorto Diagnostics, Hellerup, Denmark) (IgE-mediated and non-IgE mediated degranulation), followed by 30 min of stimulation with streptavidin peroxidase (100 ng/mL) or calcium ionophore A23187 (1 µM, Sigma) in Tyrode’s buffer containing 0.1% BSA. Total β-hexosaminidase was obtained by lysing LAD-2 cells in 0.1% Triton X-100 in PBS. The supernatants were collected and incubated with an equal volume of p-nitrophenyl N-acetyl-β-D-glucosamide (Sigma, 4 mM in citrate buffer) for 1 h. The reactions were stopped by adding 0.4 M glycine buffer, and the signals were read at 405 nM. The percentage of degranulation was calculated as 100 × (OD stimulated − OD unstimulated)/(OD total lysate − OD unstimulated).
LAD-2 cell degranulation was evaluated by the β-hexosaminidase release test44 . Cells were treated for 16 h with cis- or trans-UCA with or without simultaneous sensitisation with biotinylated-IgE (100 ng/mL, BioPorto Diagnostics, Hellerup, Denmark) (IgE-mediated and non-IgE mediated degranulation), followed by 30 min of stimulation with streptavidin peroxidase (100 ng/mL) or calcium ionophore A23187 (1 µM, Sigma) in Tyrode’s buffer containing 0.1% BSA. Total β-hexosaminidase was obtained by lysing LAD-2 cells in 0.1% Triton X-100 in PBS. The supernatants were collected and incubated with an equal volume of p-nitrophenyl N-acetyl-β-D-glucosamide (Sigma, 4 mM in citrate buffer) for 1 h. The reactions were stopped by adding 0.4 M glycine buffer, and the signals were read at 405 nM. The percentage of degranulation was calculated as 100 × (OD stimulated − OD unstimulated)/(OD total lysate − OD unstimulated).
7
Generating iPSCs from Fibroblasts
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Human fibroblasts were obtained from a 7-year-old male patient and were transduced with the CytoTune-iPS Reprogramming Kit (Life Technologies, Grand Island, NY, USA) according to the manufacturer’s instructions. Fibroblasts (2 × 104 cells/cm2) were plated into 24-well culture plates, coated with gelatin containing Stempro 34 medium (Life Technologies), and supplemented with L-ascorbic acid (S4641, Sigma-Aldrich, St. Louis, MO, USA), stem cell factor (Peprotech, Rocky Hill, NJ, USA), and bFGF (Peprotech) 7 days before transduction. Transduced cells were seeded onto mouse embryonic fibroblast (MEF)-coated plates 1 week after transduction. After 3 weeks, colonies were selected and expanded in stem MACS medium (Miltenyi Biotec, Bergisch Gladbach, Germany) on plates coated with vitronectin (VTN; Life Technologies) [19 (link)].
8
Mast Cell Activation by IgE and TIMP-1
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LAD-2 cells were provided by the National Institute of Allergy and Infectious Diseases (Bethesda, MD, USA) and cultured in StemPro-34 medium (Life Technologies) supplemented with 2 mM L-glutamine (Gibco), 1% penicillin-streptomycin, and 100 ng/mL recombinant human stem cell factor (R&D Systems). LAD-2 cells (5 × 104 cells) were stimulated with 10 ng/mL human simultaneous biotinylated-IgE (BioPorto Diagnostics, Hellerup, Denmark) with or without 100 ng/mL TIMP-1 in serum-free StemPro-34 medium plus 1% penicillin-streptomycin for 24 hours. To create cross-linking of 2 IgE molecules on LAD-2 cells, 100 ng/mL streptavidin-horseradish peroxidase conjugate having the ability to bind to biotinylated proteins was added to supernatants for 6 hours before harvesting. In some conditions, cells were pretreated with 1 µg/mL Dex for 30 minutes. Supernatants were collected for ELISA.
9
Reprogramming PBMCs into iPSCs
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PBMCs were isolated from patients’ blood by Percoll gradient separation, purified with multiple rounds of washing in DPBS, and plated in a 24-well plate. 1–2 × 106 PBMCs were cultured in 1 mL of StemPro-34 medium (Thermo Fisher) supplemented with 100 ng/mL SCF and 20 ng/mL IL-3 (Peprotech), 100 ng/mL FLT3, 20 ng/mL IL-6, and 20 ng/mL EPO (Thermo Fisher). Medium was replaced every other day until cell number stabilized. 2 × 105 PBMCs were then resuspended in 300 μl of complete PBMC medium and reprogrammed using the CytoTune®-iPSC Sendai Reprogramming Kit (Thermo Fisher). After 24 hr, media was replaced followed by every 2 days. On day 7, cells were re-plated with a 1:1 mix of Stem-MACS™ iPSC-brew XF medium with supplement (Brew) and Stem-Pro™-34 media. On d8, a full switch over to the Stem- MACS™ media was performed. Media was replaced as colonies appeared on d10–15 (Yildirim et al., 2022 (link)).
10
Efficient PBMC Isolation and iPSC Generation
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PBMCs were isolated from whole blood samples using Percoll density gradient medium (17089109, GE Healthcare). Cells were purified with multiple rounds of DPBS wash (14190144, ThermoFisher Scientific). Cells were cultured in complete PBMC medium composed of StemPro®−34 SFM medium (10639011, ThermoFisher Scientific) containing 100 ng/mL SCF (300–07, Peprotech), 100 ng/mL FLT3 (PHC9414, ThermoFisher Scientific), 20 ng/mL IL-3 (200–3, Peprotech), 20 ng/mL IL-6 (PHC0063, ThermoFisher Scientific), and 20 ng/mL EPO (PHC9631, ThermoFisher Scientific). The medium was refreshed daily until the cell count remained stable for a few days. PBMC transduction was performed using the Sendai virus reprogramming cocktail according to the manufacturer’s instructions (CytoTune™-iPSC 2.0 Sendai Reprogramming Kit, A16517, ThermoFisher Scientific). The transduced cells were resuspended and plated in a Matrigel-coated plate (356231, Corning) and cultured in StemPro™−34 medium (Thermo Fisher). Media was replaced daily until day 7 post transduction. On Day 7, the medium was switched to StemMACS™ iPS-Brew XF medium (130–104–368, Miltenyi Biotec) and was replaced every other day until Day 10–15 post transduction until the colonies appeared. Selected colonies were expanded and frozen down until experimental usage.
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