For in vitro analysis of tumor and immunogenic cell death markers, the BRCA1-mutated (insertion C at nucleotide 5382) triple-negative human cell line HCC1937 was used. The cell line was maintained in RPMI (0.6% human insulin, 10% fetal bovine serum) and authenticated using short tandem repeat profiling. Cells were treated with vehicle control (PBS), cisplatin (2 μM), recombinant human IFN-γ (BD; 5 ng/ml), or a combination of IFN-γ (5 ng/ml) and cisplatin (2 μM) for 72 hours. After this, FACS analysis was undertaken for several markers: human HLA-ABC (BD; clone G46-2.6), HLA-DR (BioLegend; clone L243), CD80 (BD; clone L307.4), CD86 (BioLegend; clone IT2.2), PD-L1 (BioLegend; clone 29E.2A3), calreticulin (Abcam; clone EPR3924), and MICA/B (BioLegend; clone 6D4). All experiments were conducted in triplicate, and three independent experiments were undertaken. All cell surface stains were incubated for 30 min at 4°C. Sample data were acquired on a Fortessa flow cytometer (BD Biosciences) and analyzed using FlowJo software (TreeStar). Data are presented as mean fluorescence index.
Recombinant human ifn γ
Manufactured by BD
Sourced in United States
Recombinant human IFN-γ is a purified protein produced through recombinant DNA technology. It is a cytokine that plays a role in immune system regulation and response.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Fortessa flow cytometer, by BD (1 mentions)
Opteia, by BD (1 mentions)
Penicillin, by Corning (1 mentions)
Streptomycin, by Corning (1 mentions)
L glutamine, by Caisson (1 mentions)
Dulbecco modified eagle medium (dmem), by Caisson (1 mentions)
Ranitidine, by Merck Group (1 mentions)
Chlorpheniramine, by Merck Group (1 mentions)
Histamine, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Leupeptin, by Merck Group (1 mentions)
Il 1β, by R&D Systems (1 mentions)
Tnf α, by R&D Systems (1 mentions)
Cellquest software, by BD (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
Midimacs, by Miltenyi Biotec (1 mentions)
Lipopolysaccharides (lps), by BD (1 mentions)
Ficoll hypaque, by GE Healthcare (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
14 protocols using recombinant human ifn γ
1
Analysis of Tumor Immunogenicity in BRCA1-mutant Cells
2
Quantifying Anti-IFN-γ Autoantibodies
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The method has been published7 (link),14 (link),20 (link),21 (link). Heparinized plasma from patients was serially diluted and incubated with recombinant human IFN-γ (BD Biosciences) at a final concentration of 300 pg/ml for 1 h at 37 °C. The level of unbound IFN-γ in the pre-incubation mixture was determined by a human IFN-γ ELISA kit (BD OptEIA: BD Biosciences), following the manufacturer’s instructions. The titer of the anti-human-IFN-γ autoantibody was determined in the highest dilution with >50% inhibition (<150 pg/ml of IFN-γ was detected).
3
PIV3 Infection and IFN-γ Treatment in A549 Cells
4
Detection and Titration of Neutralizing Antibodies
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All of our methods were based on our previous study with some modifications.[4 (link)] For the detection and titration of nAIGAs, a 96-well microplate was precoated with 100 μL of antihuman IFN-γ antibody/well (AHIGA; BD Biosciences, 1:250) and incubated overnight at 4°C. The plate was washed 3 times with phosphate buffer saline (PBS)-Tween 0.05% and then blocked by incubation with PBS-10% fetal bovine serum (Gibco) for 1 hour at room temperature (RT). In another microplate, plasma from patients and donors was serially diluted (10–1 to 10–6) and incubated with 200 pg/mL recombinant human IFN-γ (BD) for 1 hour at 37°C. After blocking, the AHIGA-precoated plate was thoroughly washed, and a mixture of recombinant human IFN-γ and diluted plasma from patients or donors (100 μL/well) was added. The plate was then incubated for 2 hours at RT and washed again, and the detection antibody (BD, 1:250) was added. After incubation for 1 hour at RT, the plate was washed 7 times, and substrate solution was added. The plate was then incubated for 5 to 10 minutes at RT. The defined titer of nAIGAs was based on the optical density value (450 nm). In the blocking assay, the plasma was diluted from 10–1 to 10–6, and the blocking index was defined as the maximum dilution of plasma that blocked 50% of the IFN-γ concentration measurement. All molecular experiments were performed in duplicate.
5
Inflammatory Cytokine Reconstitution Protocol
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Recombinant Human IFNγ (BD Biosciences, Australia), IL-1β, and TNFα (R&D Systems, Minneapolis, MN) were reconstituted in sterile PBS containing 0.1% bovine serum albumin (BSA). Histamine, chlorpheniramine, and ranitidine (Sigma-Aldrich, Sydney, Australia) were reconstituted in water for irrigation (Baxter, Sydney, Australia). Human lung tryptase (≥5,000 mU/mg/mL of vehicle consisting of 1 M NaCl, 50 mM sodium acetate, 0.01% sodium azide, and 50 μM heparin) was obtained from Calbiochem (La Jolla, CA, USA). BSA and leupeptin were purchased from Sigma-Aldrich. All agents were stored in aliquots at −20°C, except for IFNγ which was stored at −80°C.
6
Surface Marker Analysis of hFCPCs
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For the surface marker analysis, hFCPCs at passage four were untreated or stimulated with 200 U/ml of recombinant human IFN-γ (BD Biosciences, San Jose, CA, USA) for 4 days, and then incubated with fluorescence-conjugated primary antibodies against HLA-ABC, HLA-DR, CD80, CD86, CD40, CD40L, and CD11c (all from BD Biosciences) for 30 min at 4°C. After washing, the stained cells were analyzed on a BD FACSCanto II flow cytometer using Cell Quest software (BD Biosciences).
7
Isolation and Stimulation of Human Monocytes
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Human monocytes (CD14+ cell population) were isolated from peripheral blood mononuclear cells (PBMC) derived from healthy volunteers using the Ficoll Hypaque gradient (GE Healthcare Bio-Sciences AB, Sweden) method followed by positive selection using magnetic cell sorting (Midi Macs, MiltenyiBiotec, Auburn, CA, USA). Freshly purified monocytes were adjusted to 106/ml in complete RPMI-1640 medium (2mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, 10% fetal bovine serum) and distributed into 96-well tissue culture plates. Cells were stimulated either by LipESP (10μg/ml P23, LiEF-1α and LiAAA-ATPase; 5μg/ml P15), in the presence or absence of LPS (Sigma-Aldrich) at 1μg/ml; then incubated for 24 h at 37°C under 5% CO2 or primed with 3000 U/ml of recombinant human IFN-γ (BD Biosciences Pharmingen) for 12 h then stimulated with LipESP, with or without LPS and incubated for 24 additional hours. Independent experiments were run for donor’s cells. To evaluate cytokine (IL-12p70, IL-10, TNF-α) production by monocytes, 24 h and 36 h supernatants were collected and stored at –80°C until further use.
8
Quantification of Plasma IgG Binding to IFN-γ
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The method has been published11 (link),17 (link)–19 (link). In brief, recombinant human IFN-γ (BD Biosciences) was coated onto ELISA plates (Maxisorp, Nunc) at 4 °C overnight while the uncoated control wells had only PBS. On the day of the experiment, a pre-coated plate was washed, and heparinized plasma added at 1:100 dilution in duplicate, and incubated for 2 h at room temperature. After washing, biotinylated mouse anti-human IgG monoclonal antibody (clone G18–145: BD Biosciences) and horse radish peroxidase (HRP) conjugated streptavidin (BD Biosciences) was added to each well. After 1 h incubation, the color was developed by adding 3,3′,5,5′-Tetramethylbenzidine substrate (BD Biosciences). The results were calculated as the absorbance index by (O.D.test – O.D.uncoated)/O.D.uncoated.
9
Measuring IDO mRNA Expression in PBMCs
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For the measurement of IDO mRNA expression 5 × 105 freshly thawed PBMCs from trial volunteers were stimulated for 16 hours with 85A peptides or media alone. For blocking experiments PBMC were cultured with or without the addition of 10 μg/ml anti-IFN-γ or 10 μg/ml recombinant human IFN-γ (BD Biosciences). CD14+ cells were depleted from buffy coat PBMC from PPD+ donors using CD14 labeled magnetic beads (Invitrogen).
10
Quantifying Phospho-STAT1 in Myeloid Cells
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The method has been published14 (link),19 (link). Heparinized plasma was diluted at 1:10 and incubated with and without 200 ng/ml recombinant human IFN-γ (BD Biosciences) at 37 °C for 1 h. Frozen human peripheral blood mononuclear cells (PBMCs) were thawed, adjusted to 1 ×105 cells/ml, and labeled with fluorescein isothiocyanate (FITC) conjugated mouse anti-human CD14 monoclonal antibody (clone M5E2: BD Biosciences) at room temperature for 30 min in the dark. FITC-CD14 labeled PBMCs were aliquoted to sterile tubes and incubated with of pre-incubated heparinized plasma at 37 °C for 30 min. Cells were washed thrice and fixed with 2% paraformaldehyde at room temperature for 15 min. After washing thrice, ice-cold 90% methanol was added and incubated on ice for 40 min. After washing thrice, phycoerythrin (PE) Mouse anti-human phospho-STAT1 (pY701) monoclonal antibody (clone 4a: BD Biosciences) was added and incubated on ice for 1 h before washing thrice. The population of FITC-CD14 positive with and without PE-pSTAT1 was analyzed by flow-cytometer with FlowJo version 10 software (Supplementary Fig. S1 ). Results are shown as histograms or calculated median fluorescent intensity (MFI) into % pSTAT1 stimulation index, as following:
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