Data represented in Figures 5 A–5L: Cytokines were added for 12 h overnight at increasing doses (0, 0.1, 0.5, 1, 2, 5, 10 ng/mL) of IL-4 (human: Biolegend 574002), IL-17A (human: Biolegend 570502), IFNγ (human: Biolegend 570202; mouse: Peprotech 315-05), IFNα (human: Biolegend 592702; mouse: Biolegend 752802), or IFNβ (mouse: R&D Systems 8234-MB-010). Each condition was run as a biological triplicate. Data represented in Figure S3 C-K: cytokines were added for 12 h overnight at increasing doses (0, 0.1, 0.5, 1, 5, 10 ng/mL) of human IL-4 (Biolegend 574004), IL-13 (Biolegend 571104), IFNα (Biolegend 592704), IFNγ (Biolegend 570204), IL-17A (Biolegend 570504), or IL-1β (Biolegend 579404) (each condition run as a biological quadruplicate). All populations were lysed in 50 μL lysis buffer (RLT + 1% BME, QIAGEN and Sigma, respectively) and snap frozen on dry ice. Bulk RNA-seq was performed as described previously and summarized above (Ordovas-Montanes et al., 2018 (link)). Populations were sequenced to an average ± SEM read depth of 3.95 ± 0.11 million reads per sample, with an average ± SEM alignment percentage to either hg19 or mm10 reference transcriptomes of 71 ± 0.3%. All samples met quality thresholds regarding genomic and transcriptomic alignment.
Ifn α
Manufactured by BioLegend
Sourced in United States
IFN-α is an interferons protein produced by various cell types in response to the presence of pathogens. It serves a core function in the immune system's antiviral response.
Automatically generated - may contain errors
Lab products found in correlation
Ifn γ, by BioLegend (12 mentions)
Ifn β, by BioLegend (5 mentions)
Interleukin 4 (il 4), by BioLegend (4 mentions)
Interleukin 6 (il 6), by BioLegend (4 mentions)
Il 1β, by BioLegend (3 mentions)
Ifn γ, by Thermo Fisher Scientific (3 mentions)
Il 13, by BioLegend (2 mentions)
Il 17a, by BioLegend (2 mentions)
Ifn β, by R&D Systems (2 mentions)
Il 27, by BioLegend (2 mentions)
Fix perm cell permeabilization kit, by Thermo Fisher Scientific (2 mentions)
Streptavidin allophycocyanin, by Thermo Fisher Scientific (2 mentions)
Cytoflex s, by Beckman Coulter (2 mentions)
Anti gfp ab, by Thermo Fisher Scientific (2 mentions)
Anti cd8 biotin ab, by Thermo Fisher Scientific (2 mentions)
Il 21, by BioLegend (2 mentions)
Il 23, by BioLegend (2 mentions)
Gm csf, by Thermo Fisher Scientific (2 mentions)
Il 12, by BioLegend (2 mentions)
Ly294002, by Merck Group (2 mentions)
23 protocols using ifn α
1
Dose-Dependent Cytokine Profiling for Transcriptomic Analysis
2
Zbtb20 Expression in Cultured Splenic B Cells
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AutoMACS-isolated splenic B cells from naïve mice were cultured in the presence of IL-1β (5 ng/mL; BioLegend), IL-6 (40 ng/mL; BioLegend) IL-9 (12.5 ng/mL; BioLegend), IL-12 (5 ng/mL; R&D Systems), IL-33 (40 ng/mL; Peprotech), IFN-α (1000 U/mL; BioLegend), IFN-β (500 U/mL; BioLegend), or IFN-γ (200 ng/mL; BioLegend) with lipopolysaccharide (LPS; 10 ng/mL) for 24 h at 37 °C. Cells were subjected to Zbtb20 intracellular staining or lysed for qRT-PCR assays.
3
Immortalized Mouse Macrophage Reporter Cell Line
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Immortalized bone marrow-derived mouse macrophage (iBMDM) cells (37 (link)) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with L-glutamine (Sigma-Aldrich), supplemented with 10% (v/v) of heat-inactivated Foetal Bovine Serum (Life Technologies Ltd) and 1% (v/v) of Penicillin-Streptomycin (Sigma-Aldrich) solution. Cells were cultured between passages 6-30 in sterile NuncTM 10 cm cell culture petri dishes (ThermoFisher Scientific) till 80-90% confluent. Lentiviral transduction (38 (link)) was used to develop reporter iBMDM line expressing murine STAT1 coding sequence C-terminally fused with the red fluorescent protein (STAT1-tagRFP) and murine STAT6 coding sequence N-terminally fused with yellow fluorescent protein (Venus-STAT6). Additionally, cells expressed histone H2B protein fused with cyan fluorescent protein (AmCyan-H2B). iBMDMs were sequentially transduced and triple positive cells were identified using fluorescence single cell sorter (BD Influx) to derive a clonal reporter line. Cells were stimulated with recombinant mouse IFN-γ (575306, Biolegend), IL-4 (574306, Biolegend), IFN-α (752802, Biolegend) and IFN-β1 (581302, Biolegend).
4
In Vitro NHEK Culture and Stimulation
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Normal human epidermal keratinocytes (NHEKs) were purchased from Thermo Fisher Scientific and maintained for up to 6 passages in T-75 flasks or used earlier. Cells were grown in serum-free EpiLife cell culture medium with EpiLife Defined Growth Supplement containing 0.06 mM Ca2+ (Gibco, Waltham, MA) or Keratinocyte serum-free-media (KSFM) with supplements provided by manufacturer (Gibco) and additional 0.06mM Ca2+. NHEKs were grown to approximately 75-80% confluence. For experiments, cells between passage 3-6 were plated at approximately 200,000 cells/well in 6-well plates and 75,000 cells/chamber in 2-chamber slides, respectively (LabTek, Bloomington, IN). For some experiments, IL-27 was used at a concentration of 100 ng/mL; IFN-α was used at a concentration of 50 U/mL (BioLegend). The cells were collected for quantitative RT-PCR or immunofluorescence at various time points.
5
Cytokine-Induced STAT Phosphorylation Assay
6
Cytokine-Induced Keratinocyte Response
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Medium was removed, and new medium containing cytokines was used to stimulate keratinocytes for a variety of time points. Unless stated otherwise, IL-6, IL-23, IL-27, IL-35 (all from BioLegend), and IL-12 (Tonbo) were added to 1 to 2 ml of medium at a concentration of 100 ng/ml; IFN-α (BioLegend) or IFN-γ (PeproTech) was added to 1 to 2 ml of media at a concentration of 50 U/ml. For the cytotoxicity study, cells were suspended in CellTiter 96 AQueous One Solution Reagent and then incubated at 37°C for 1 to 4 hours in a humidified, 5% CO2 atmosphere. Absorbance was read at 490 nm using a 24-well plate reader.
7
Lupus Murine Model Analysis
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MRL/lpr-Igkm/h mice have been previously described (8 (link), 9 (link)). Male and female mice 6-16 weeks of age were used in experiments. For some analyses, groups of 6-7 week old MRL/lpr-Igkm/h mice were first injected i.p. with 200 μg of CpG ODN 1826 (IDT) twice a week for four weeks, or with 0.9 μg or 9.0 μg of IFNα (BioLegend) every other day for one week. Control mice were injected with equal volume of PBS. Mice were housed in Specific Pathogen Free conditions at the University of Colorado Anschutz Medical Campus (UCD-AMC). All animal procedures were approved by the UCD-AMC Institutional Animal Care and Use Committee and carried out in accordance with approved guidelines.
8
Cytokine-Induced STAT Phosphorylation Assay
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Forty-eight hours after transduction, cells were first reseeded in IL-2–free media for 4 h, then cultured in RPMI medium with 10% FBS and anti–CD8-biotin Ab (53–6.7; eBioscience) in the presence or absence of rIL-2 (50 U/ml) for 10 min to induce STAT5 phosphorylation. For STAT1 and STAT3 phosphorylation, cells were cultured in the presence or absence of IFN-α (5000 U/ml; 30 min; BioLegend) or IL-21 (200 ng/ml; 30 min; BioLegend), respectively, and anti–CD8-biotin was added for the last 10 min. Cells were then fixed in 2% paraformaldehyde (5 min) and fixation reagent (15 min) (FIX & PERM Cell Permeabilization Kit; Thermo Fisher Scientific) and made permeable with ice-cold methanol (15 min) and permeabilization medium (FIX & PERM Cell Permeabilization Kit; Thermo Fisher Scientific), then stained with anti-GFP Ab (Thermo Fisher Scientific), anti–p-STAT1-PE (A15158B), anti–p-STAT3-AF647 (13A3–1; all from BioLegend), anti–p-STAT5 allophycocyanin (SRBCZX), streptavidin-PE (all from eBioscience), and streptavidin-allophycocyanin (Invitrogen). Cells were analyzed with a CytoFLEX S (Beckman Coulter) Flow Cytometer.
9
Generating GM-BMDMs and CD122+ Macrophages
10
Stimulation and Activation of BMDMs
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For TLR4 stimulation, BMDMs were stimulated with different doses of LPS (Invivogen) for 30 min, washed, and further incubated for 5.5 h before harvest. To evaluate BMDMs' response to poly(I:C) via endosomal/lysosomal pathway or cytosolic PPR sensing pathway, BMDMs were either incubated with 50 μg/ml of naked poly(I:C) or transfected with 2 μg/ml of Lipofectamine 2000 (Invitrogen) packaged poly(I:C) (Invivogen), respectively. For cytokine stimulation, BMDMs were treated with different doses of IFN‐α (BioLegend), IFN‐β (R&D systems), and IFN‐γ (BioLegend) for various length of time as indicated in the figures.
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