A ten-plex assay was set up in order to simultaneously measure 10 different cytokines in the plasma. For granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage-CSF (GM-CSF), interleukin-3 (IL-3), IL-6 and mastocyte-CSF (M-CSF) detection, commercially available beads were used (Bio-Rad, Marnes la coquette, France). For erythropoietin (EPO), stem cell factor (SCF), stromal cell derived factor-1 (SDF-1), FMS like tyrosine kinase 3 ligand (Flt3-l) and thrombopoietin (TPO) specific beads were developed using specific antibodies (all from R&D system, Abingdon, UK), uncoupled beads and a coupling kit (All from Bio-rad) according to the manufacturer’s recommendations. The range in which a linear response is obtained, specificity and absence of cross-reactivity of these beads were then assessed using recombinant mouse cytokines (all from R&D systems) before mixing the ten beads in a single assay. Detection limits, defined as 2σ above the mean blank control value were 0.005 ng/ml for EPO, GM-CSF, IL-3, M-CSF and Flt3-l, 0.01 ng/ml for G-CSF and IL-6, and 0.025 ng/ml for SCF, TPO and SDF-1.
Recombinant mouse cytokines
Manufactured by R&D Systems
Sourced in United States
Recombinant mouse cytokines are proteins produced in a laboratory setting that are derived from mice. These cytokines function as signaling molecules and play a role in cellular communication and immune response.
Automatically generated - may contain errors
Lab products found in correlation
Macs lineage cell separation kit, by Miltenyi Biotec (2 mentions)
B220 apc and cd11b apc cy7 antibodies, by BioLegend (2 mentions)
Trizol ls, by Thermo Fisher Scientific (2 mentions)
Specific antibodies, by R&D Systems (1 mentions)
Coupling kit, by Bio-Rad (1 mentions)
M csf, by Bio-Rad (1 mentions)
Anti cd3 and anti cd28 antibody, by Thermo Fisher Scientific (1 mentions)
Ova323 339, by AnaSpec (1 mentions)
Macs bead, by Miltenyi Biotec (1 mentions)
Diff quik stain set, by Baxter (1 mentions)
Fitc conjugated anti mouse cd4 clone gk1.5, by Thermo Fisher Scientific (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Taurine, by Merck Group (1 mentions)
Non essential amino acids (neaa), by Merck Group (1 mentions)
2 3 butanedione monoxime, by Merck Group (1 mentions)
Hepes, by Quality Biological (1 mentions)
Protease xiv, by Merck Group (1 mentions)
Penicillin streptomycin, by Quality Biological (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Collagenase 2, by Merck Group (1 mentions)
11 protocols using recombinant mouse cytokines
1
Multiplex Cytokine Quantification Assay
2
In Vitro T Cell Differentiation
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Total splenocytes or naive CD4+/CD62L+ cells were incubated for 3 days in the presence of IL-2 (50 units/mL), IL-12(10 ng/mL), and anti-IL4(5 μg/mL) for Th1 cell differentiation; IL-2 (50 units/mL), IL-4 (10 ng/mL), and anti-IFNγ (5 μg/mL) for Th2 cell differentiation; or IL-6(40 ng/mL), TGFβ(5 ng/mL), IL-23(10 ng/mL), anti-IL4(5 μg/mL) and anti-IFNγ (5 μg/mL) for Th17 cell differentiation. All recombinant mouse cytokines were purchased from R&D Systems, Inc (San Diego, CA) and eBioscience. OT-II and DO11.10 splenocytes were activated by chicken ovalbumin peptide, OVA (323–339), (AnaSpec, Fremont, CA) and naïve CD4+/CD62L+ T cells were activated by plate-bound anti-CD3 and anti-CD28 antibodies from eBioscience, Inc. Naïve CD4+/CD62L+ T cells were isolated by MACS beads from Miltenyi Biotec (Auburn, CA).
3
Intranasal OVA Challenge Induces Airway Hyperreactivity
4
Lineage Depletion and Cell Culture Protocol
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Nucleated cells from the femur and tibia of 3-week post-pIpC mice were lineage depleted with a MACS lineage cell separation kit according to manufacturer’s instructions (Miltenyi Biotec, Auburn, CA). Lineage depleted cells were cultured onto 30,000 OP9 cells (plated night before) in IMDM supplemented with 10% defined FBS, 55mM BME, 50 U/ml penicillin, 50mg/mL streptomycin, 0.1mM Glutamax, 10ng/mL Flt3L, and 5ng/mL IL-7. Recombinant mouse cytokines were obtained from R&D Systems (Minneapolis, MN) or Invitrogen (Carlsbad, CA). Cells were transferred onto fresh OP9 cells every 3 days. To evaluate myeloid and B cell differentiation, cells were analyzed 6 days after culture with B220-APC and CD11b-APC/cy7 antibodies (BioLegend, San Diego, CA).
5
Cytokine-Driven T Cell Activation
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Recombinant mouse cytokines were obtained from R&D Systems. Propidium Iodide (PI) was obtained from Sigma-Aldrich. FITC-conjugated anti-mouse CD4 (clone GK1.5), APC-conjugated anti-CD8 (Ly-2) and PE-conjugated anti-CD25 (PC61) mAbs were purchased from eBioscience. Purified anti-CD3ε (145-2C11), anti-CD28 (37.51), anti-mouse-IL-2(S4-B6) and anti-mouse Bcl2 staining kit were purchased from BD Biosciences. Mitotracker Red staining kit to measure mitochondrial membrane potential and anti-CD3/CD28 coated beads were purchased from Molecular Probes (Invitrogen).
6
Murine Hematopoietic Cell Transduction
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Wildtype and mirn23a-/- C57BL/6 mice were intraperitoneally injected with 5mg 5-fluorouracil in 100 uL 1X phosphate buffered saline (PBS). Bone marrow was harvested 3 days later and red blood cells were removed by ammonium chloride lysis. Nucleated blood cells were cultured in IMDM supplemented with 20% horse serum, 10% HM3 conditioned media (as a source of GM-CSF), 20ng/mL human IL-6, and 10 ng/mL murine IL-1b. Recombinant mouse cytokines obtained from R&D Systems (Minneapolis, MN, USA) or Invitrogen. Cells were then spin transduced with RARα403 from supernatants of GP + E86 producer lines for 2 hours at 32 degrees Celsius at 3200 RPM with 5ug/mL polybrene. Following spin transduction, cells were transferred to IMDM supplemented with 20% horse serum, 10% KSL CM (as a source of SCF), 10% WEHI-3B (as a source of IL-3), and 8 U/mL human erythropoietin. Cells were then passaged every 2–3 days in IMDM supplemented with 20% horse serum, 10% COS-KSL (as a source of SCF). Cells were selected for neomycin resistance by treating cells with G418 for 10 days and continuing cultures with the live cells. EML phenotype was confirmed by flow cytometry.
7
Isolation and Culture of Mouse Cardiac Fibroblasts
8
In vitro Expansion and Manipulation of ILC2s
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Sorted ILC2s were cultured (37°C, 10% CO2) at 3,000–10,000 cells per well in 200 µl high-glucose DMEM supplemented with 10% heat-inactivated FBS, penicillin/streptomycin/l -glutamine, 2-mercaptoethanol, recombinant mouse cytokines, IL-33, TSLP, and IL-7 at 10 ng/ml (R&D Systems). At harvest, cell-free culture supernatants were collected, and cells were analyzed by flow cytometry or collected in TRIzol LS (Thermo Fisher Scientific) for RNA extraction. On day 8 of culture, ILC2s were transfected with miRNA mimics and siRNAs (Dharmacon). miRIDIAN miRNA mimics were used at 500 nM per transfection. siGENOME SmartPools and ON-TARGETplus Individual siRNAs were used at 500 nM. Cells were transfected using the Neon Transfection system (Invitrogen) as previously described (Steiner et al., 2011 (link)). Cells and culture supernatants were harvested on day 10 for further analysis.
9
Cell Culture Conditions for Various Cell Lines
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70Z/3 cells (ATCC, Manassas, VA) were grown in RPMI supplemented with 10% FBS, 0.1 mM glutamax, 10 mM HEPES, and 1 mM sodium pyruvate. 293FT cells (Invitrogen, Carlsbad, CA) were grown in OptiMEM supplemented with 5% FBS. OP9 cells (ATCC, Manassas, VA) were cultured in alpha MEM supplemented with 20% FBS, 1mM sodium pyruvate, and 55uM BME. BCL1, a Balb/c derived B cell lymphoma cell line [11 (link),12 (link)], was cultured in RPMI supplemented with 10% FBS. Primary mouse cells were cocultured on OP9 cells in IMDM supplemented with 10% defined FBS, 55uM BME, 0.1mM Glutamax, 50ng/mL SCF, 10ng/mL IL-3, and 10ng/mL IL-6. Recombinant mouse cytokines were obtained from R&D Systems (Minneapolis, MN) or Invitrogen (Carlsbad, CA). All media contained 50 U/ml penicillin and 50mg/ml streptomycin. 70Z/3 RFP and 70Z/3 RFP Arid3b overexpressing cells were generated as described previously [13 (link)].
10
Cell Culture Protocols for A20, EML, and 32Dcl3
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The A20 and EML cell lines were obtained from American Type Culture Collection (Manassas, VA, USA). 32Dcl3 was a gift from Allan Friedman (Johns Hopkins University, Baltimore, MD, USA). Unless stated otherwise, cell culture media and additives were obtained from Thermo Fisher Scientific Life Sciences. A20 cells were cultured in RPMI, supplemented with 10% FBS and 55 mM 2-ME. EML cells were cultured in IMDM, supplemented with 20% FBS and 10% COS-KSL conditioned media (as a source of SCF). 32Dcl3 cells were grown in IMDM, supplemented with 10% FBS, 10% Wehi-3B conditioned media, and 55 mM 2-ME. A generation of cell lines overexpressing mirn23a miRNAs was described previously [27] . Primary mouse cells were cultured in IMDM, supplemented with 10% defined FBS, 55 mM 2-ME, 0.1 mM GlutaMAX, 50 ng/ml SCF, 10 ng/ml IL-3, and 10 ng/ml IL-6. Recombinant mouse cytokines were obtained from R&D Systems (Minneapolis, MN, USA) or Thermo Fisher Scientific Life Sciences. All media contained 50 U/ml penicillin and 50 mg/ml streptomycin.
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