Monocytes were enriched from PBMCs using CD14 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and magnetic-activated cell sorting (MACS), and then cultured in 96-well flat-bottomed plates in RPMI 1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum, 2 mM L-glutamine, 50 U/ml penicillin, 50 µg/ml streptomycin, and 55 µM 2-mercaptoethanol (all from Thermo Fisher Scientific) at 37°C in a 5% CO2 incubator. Monocytes were stimulated with recombinant human IL-3 (200 ng/ml; PeproTech, Rocky Hill, NJ, USA) and 2’3’-cGAMP (50 µg/ml; In vivoGen, San Diego, CA, USA) for 5 h, except for the immunofluorescence experiment where monocytes were stimulated for 3 h. HC monocytes used for RNA-seq experiments were pretreated with IFNα (100 U/ml) (R&D Systems, Minneapolis, MN, USA) for 18 h prior to stimulation with 2’3’-cGAMP.
Recombinant human il 3
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
Recombinant human IL-3 is a cytokine that plays a role in the regulation of hematopoiesis. It is produced by activated T cells and mast cells, and it acts on various cell types, including hematopoietic progenitor cells, mast cells, and eosinophils.
Automatically generated - may contain errors
Lab products found in correlation
Recombinant human scf, by Thermo Fisher Scientific (4 mentions)
Penicillin, by Thermo Fisher Scientific (4 mentions)
Streptomycin, by Thermo Fisher Scientific (4 mentions)
Recombinant human il 6, by Thermo Fisher Scientific (3 mentions)
L glutamine, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Penicillin, by Merck Group (2 mentions)
Streptomycin, by Merck Group (2 mentions)
Rpmi 1640 medium, by Merck Group (2 mentions)
L glutamine, by Merck Group (2 mentions)
Recombinant human tpo, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Rhil 6, by Thermo Fisher Scientific (2 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
2 3 cgamp, by InvivoGen (1 mentions)
Ifn α, by R&D Systems (1 mentions)
Cd14 microbead, by Miltenyi Biotec (1 mentions)
Basal medium eagle, by Thermo Fisher Scientific (1 mentions)
Cpg odn 2216, by Miltenyi Biotec (1 mentions)
Imiquimod r837, by InvivoGen (1 mentions)
21 protocols using recombinant human il 3
1
Monocyte isolation and stimulation protocol
2
Cytokine Profiling of Activated PBMCs
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PBMCs were cultured in 96-well flat-bottom plates in Basal Medium Eagle (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 50 U/mL penicillin, and 50 μg/mL streptomycin (all from Thermo Fisher Scientific). PBMCs were stimulated with recombinant Human IL-3 (100 ng/mL; PEPROTECH, Rocky Hill, NJ, USA) and a TLR7 agonist, imiquimod (R837) (100 ng/mL; InvivoGen, San Diego, CA, USA) or a TLR9 agonist, CpG ODN 2216 (5 μg/mL; Miltenyi Biotec, Bergisch Gladbach, Germany) for 6 h at 37 °C in a 5% CO2 incubator. GolgiPlug (100 ng/mL; BD Biosciences) was added during the final 3 h of stimulation to block cytokine secretion. After staining the cell-surface antigens, intracellular cytokines were stained using the BD Cytofix/Cytoperm Fixation/Permeabilization Solution Kit (BD Biosciences), anti-IFN-α-APC (Miltenyi Biotec), and anti-tumor necrosis factor α (TNF-α)-PE-Cy7 (BD Biosciences), or their isotype control antibodies.
3
Comprehensive Western Blot and Flow Cytometry Protocol
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Antibodies against ZNF384 (Cat. Ab176689; Abcam, Cambridge, UK), p-STAT5 (Cat. 9314; CST, Danvers, MA, USA), STAT5 (Cat. 94,205; CST), FLAG (Cat. F1804; Merck, Darmstadt, Germany), and GAPDH (Cat. 60004-1-Ig; Proteintech Group, Rosemont, IL, USA) were used for western blot analysis. The following antibodies were used for flow cytometry analysis: APC mouse anti-human CD123 (Cat. 560087; BD Biosciences, Franklin Lakes, NJ, USA), APC mouse IgG2a, κ Isotype Control (Cat. 555576; BD Biosciences), APC Mouse Anti-Human CD19 (Cat. 340437; BD Biosciences), and an IgG1 kappa isotype control (Cat. 340442, BD Biosciences). The doxycycline hyclate was purchased from Meilunbio (Cat. MB1088; Dalian, China). Recombinant human IL-3 was purchased from PeproTech (Cat. 200-03; Cranbury, NJ, USA). Doxorubicin hydrochloride (Cat. T1020), dexamethasone (Cat. T1076) and vincristine sulfate (Cat. T1270) were purchased from TOPSCIENCE (Singapore). Puromycin was purchased from Selleck Chemicals (Cat. S7417; Houston, TX, USA). Enzyme-linked immunosorbent assay (ELISA) kit for IL-3 was purchased from Cloud-Clone Corp. (Cat. SEA076Hu; Houston, TX, USA). Pegaspargase was purchased from Jiangsu Hengrui Pharmaceutical Co., Ltd.
4
Hematopoietic Cell Culture Reagents
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Saracatinib (Cat# S1006), vemurafenib (Cat# S1267), rapamycin (Cat# S1039) and hemin (Cat# S5645) were purchased from Selleck Chemicals (Shanghai, China). Recombinant human SCF (Cat# 255-SC-200) was purchased from R&D Systems (Shanghai, China). Recombinant human IL-3 (Cat# GMP200-03) was purchased from Peprotech (Suzhou, China). Erythropoietin (EPO) was purchased from Darbepoetin alfa Administration (Kyowa Hakko Kirin, Japan). StemSpan SFEM (Cat# 09650) was purchased from STEMCELL Technologies. Propylhydrazine hydrochloride (Cat# 114,715) was purchased from Sigma Aldrich.
5
Isolation and Culture of Primary Human Plasmacytoid Dendritic Cells
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Primary pDCs were isolated from human heparinized leukocyte-enriched buffy coats, which were obtained from healthy blood donors drawn at the Regional Blood Center of Hungarian National Blood Transfusion Service (Debrecen, Hungary) in accordance with the written approval of the Director of the National Blood Transfusion Service and the Regional and Institutional Ethics Committee of the University of Debrecen, Faculty of Medicine (Debrecen, Hungary). Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque (GE Healthcare, Uppsala, Sweden) density gradient centrifugation. Plasmacytoid DCs were then purified from PBMCs by positive selection using the human CD304 (BDCA-4/Neuropilin-1) MicroBead Kit (Miltenyi Biotec, Bergish Gladbach, Germany), according to the manufacturer's protocol. After separation on VarioMACS magnet, the purity of isolated pDCs was > 96% as confirmed by flow cytometry.
Freshly isolated pDCs were cultured in 48-well cell culture plates at a density of 5 × 105 cells/ml in RPMI 1640 medium (Sigma-Aldrich) supplemented with 10% heat-inactivated FBS (Life Technologies Corporation), 2 mML -glutamine, 100 U/ml penicillin, 100 ng/ml streptomycin (all from Sigma-Aldrich), and 50 ng/ml recombinant human IL-3 (Peprotech EC, London, UK). During treatments cells were incubated at 37 °C in a humidified atmosphere containing 5% CO2.
Freshly isolated pDCs were cultured in 48-well cell culture plates at a density of 5 × 105 cells/ml in RPMI 1640 medium (Sigma-Aldrich) supplemented with 10% heat-inactivated FBS (Life Technologies Corporation), 2 mM
6
Cell Culture Conditions for Myeloid Leukemia
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TF-1a, TF-1, HEL, and THP-1 AML cell lines were cultured in Roswell Park Memorial Institute (RPMI)1640 (Biowest, France), supplemented with 10% fetal bovine serum (Biowest) and 1% of 100× penicillin/streptomycin (P/S) (Biowest) at 37 °C with 5% CO2. Additional 4 ng/μl of recombinant human IL-3 (Preprotech, NJ, USA) was also added for TF-1. HEK-293T cells were maintained in Dublecco’s modified Eagle’s medium (DMEM) (Biowest) supplemented with 10% FBS and 1% of P/S at 37 °C with 5% CO2.
7
Culturing Hematopoietic Cell Lines and Primary JMML Cells
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Hematopoietic cell lines PEER, K-562, MONO-MAC-6, HL-60, OCI-AML3, JURKAT, DAUDI, KASUMI-1, LOUCY, MV-4-11, and THP-1 were available in house, DEL and JVM-2 were purchased at the DSMZ repository (Braunschweig, Germany), and KARPAS-422 at Sigma-Aldrich (Saint Louis, Missouri, USA). Cell lines were grown in RPMI medium (Invitrogen, Waltham, MA, USA) supplemented with 10% or 20% Fetal Calf Serum (FCS, ThermoFisher Scientific, Waltham, MA, USA), according to supplier instructions, together with 100 U/mL Penicillin/Streptomycin (10,000 U/mL, Invitrogen) and 100 µg/mL l -glutamine (200 mM, Invitrogen). For THP-1, medium was additionally supplied with 0.05 mM β-mercaptoethanol. Cell lines were incubated at 37 °C in 5% CO2 incubators.
Mononuclear cell preparations derived from spleen and PB from 3 JMML patients were primary cultured in StemSpan SFEM II medium (Stemcell Technologies, Vancouver, Canada) supplemented with recombinant human IL3 (0.01 µg/mL; PeproTech, London, UK), FLT3L (0.01 µg/mL; PeproTech, London, UK), TPO (0.01 µg/mL; PeproTech, London, UK) and SCF (0.025 µg/mL; PeproTech, London, UK) and incubated at 37 °C in 5% CO2 incubators.
Mononuclear cell preparations derived from spleen and PB from 3 JMML patients were primary cultured in StemSpan SFEM II medium (Stemcell Technologies, Vancouver, Canada) supplemented with recombinant human IL3 (0.01 µg/mL; PeproTech, London, UK), FLT3L (0.01 µg/mL; PeproTech, London, UK), TPO (0.01 µg/mL; PeproTech, London, UK) and SCF (0.025 µg/mL; PeproTech, London, UK) and incubated at 37 °C in 5% CO2 incubators.
8
Erythroid Differentiation of Transfected CD34+ HSPCs
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Transfected CD34+ HSPCs were differentiated into mature RBCs using a three-phase erythroid differentiation protocol, as previously described12 (link),65 (link). During the first phase (day 0 to day 6), cells were cultured in a basal erythroid medium supplemented with 100 ng/ml recombinant human SCF (PeproTech), 5 ng/ml recombinant human IL-3 (PeproTech), 3 IU/ml EPO Eprex (Janssen-Cilag) and 10−6 M hydrocortisone (Sigma). During the second phase (day 6 to day 9), cells were co-cultured with MS-5 stromal cells in the basal erythroid medium supplemented with 3 IU/ml EPO Eprex (Janssen-Cilag). During the third phase (day 9 to day 20), cells were co-cultured with stromal MS-5 cells in a basal erythroid medium without cytokines. Heat-inactivated human AB serum was added during the third phase of the differentiation (10%; day 13 to day 20). Erythroid differentiation was monitored by flow cytometry analysis of CD36, CD71, GYPA, BAND3, and α4-Integrin erythroid surface markers and of enucleated cells using the DRAQ5 double-stranded DNA dye. 7AAD was used to identify live cells. The gating strategy used to assess erythroid surface markers and enucleated cells is shown in Supplementary Fig. 19 .
9
Activation of Basophils by Anti-PEG Antibodies
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Blood from healthy volunteers was collected in sodium heparin tubes, and washed in PBS. Anti-PEG or control mAbs (CH65, an anti-influenza HA mAb) were diluted in RPMI-1640/10% FBS containing recombinant human IL-3 (Peprotech), and added to blood for a final concentration of 10 μg/ml of antibody and 2 ng/ml IL-3. For positive controls, blood samples were treated with 2 μg/ml rabbit anti-human IgE (Bethyl) or LPS (O111:B4, Sigma-Aldrich) and recombinant GM-CSF (Peprotech). Cells were incubated at 37°C/5% CO2 for 30 minutes. To expose blood cells to clinical material, a volume of MPER peptide liposomes containing 10 μg/mL PEG was added to wells, and incubated for 30 minutes at 37°C/5% CO2. After the incubation, EDTA was added to a final concentration 2mM. Cells were then labeled with optimized concentrations of the following fluorochrome-mAb conjugates: FITC CD63 (clone H5C6, BD Biosciences), PE CD203c (clone NF4D6, BD Biosciences), PE-Cy7 CD123 (clone 6H6, Biolegend), APC CCR3 (clone 5E8, BD Biosciences), Alexa Fluor 700 HLA-DR (clone I243, Biolegend), BV711 CD15 (clone W6D3, Biolegend). Aqua Live/Dead (Thermo) identified dead cells. Red blood cells were lysed with FACS Lysing Buffer (BD Biosciences). Cells were analyzed on a BD LSRFortessa cytometer running Diva 8. Data were analyzed using FlowJo 10 (BD Biosciences).
10
Cell Culture Conditions for Cell Lines
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