iPSC-MSCs were seeded on fibronectin- and collagen-coated plates. The next day they were licensed with 50 ng/mL of Recombinant Human IFNγ (BioLegend, San Diego, CA) for 24 and 48 h in M-SFEM, at 37 °C in 5% CO2. Cells were split and treated as follows: RNA was isolated using the QuickRNA miniprep kit (Zymo Research, Irvine, CA) and qPCR was performed using the SYBR Green Master Mix (BioTools, Jupiter, FL) to assess indoleamine 2,3-dioxygenase (IDO) mRNA levels. β-Actin was the reference gene used for normalization. Cells were also stained with antibodies specific for surface markers, as indicated. Data were acquired on a BD LSR Fortessa Flow Cytometer (Becton Dickinson, Franklin Lakes, NJ) and analyzed using FlowJo (version 10.0, Treestar, Ashland, OR).
Recombinant human ifn γ
Manufactured by BioLegend
Sourced in United States
Recombinant human IFN-γ is a cytokine produced in E. coli. It is a protein important in immune response and inflammation.
Automatically generated - may contain errors
Lab products found in correlation
Lsrfortessa flow cytometer, by BD (2 mentions)
Quick rna miniprep kit, by Zymo Research (2 mentions)
7 aad, by BioLegend (2 mentions)
Probenecid, by Thermo Fisher Scientific (1 mentions)
Black 96 well plates with clear bottom, by Corning (1 mentions)
Sirna buffer, by Horizon Discovery (1 mentions)
L tryptophan trp, by Merck Group (1 mentions)
Liveblazer fret b g loading kit, by Thermo Fisher Scientific (1 mentions)
Phenol red free dmem, by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
96 well plate, by BD (1 mentions)
Bradford protein quantification assay, by Bio-Rad (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Chemidoc mp, by Bio-Rad (1 mentions)
Quantity one 1 d analysis software, by Bio-Rad (1 mentions)
Epacadostat, by Targetmol (1 mentions)
Ch223191, by Merck Group (1 mentions)
Mycoalert, by Lonza (1 mentions)
Recombinant human ifn β, by Thermo Fisher Scientific (1 mentions)
Recombinant human ifn λ2, by Thermo Fisher Scientific (1 mentions)
15 protocols using recombinant human ifn γ
1
IFNγ-Induced IDO Expression in iPSC-MSCs
2
High-throughput Tryptophan Metabolism Assay
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Black 96-well plates with clear bottom were purchased from Corning costar (Cat. 3904). Dharmafect (T-2001), 5X siRNA buffer (Cat. B-002000-UB-100) and siGENOME SMARTpool siRNAs as listed in Table 2 , were purchased from Dharmacon. Recombinant human IFNγ was obtained from Biolegend (Cat. 570206). L-tryptophan (Trp) and 1-Methyl L-tryptophan (1-Methyl Trp) were obtained from Sigma-Aldrich (Cat. T0254, 447439). Trp was re-suspended in Tissue Culture grade water and used at 0.3125mM unless otherwise indicated. 1-Methyl Trp was resuspended in 0.1N NaOH and used at 0.2mM. Phenol-red free DMEM and probenecid were obtained from Thermo Fisher Scientific (Cat. 21063029 and P36400). LIVEBLAzer™-FRET B/G loading kit which includes the fluorescent substrate CCF4-AM was purchased from Invitrogen (Cat. K1095).
3
IFNγ-Induced IDO Expression in iPSC-MSCs
4
Profiling PBMC Subsets upon Activation
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Young and elderly PBMCs (which includes myeloid (m)DC1, mDC2 and plasmacytoid (p)DC subsets) were cultured at 37°C in 96-well plates (BD), at 200 μl per well, in: (i) complete medium only (unstimulated control), or (ii) complete medium supplemented with DC activation stimuli: 1 μg/ml LPS (Sigma-Aldrich) and 20 ng/ml recombinant human IFN-γ (Biolegend, USA) and blood DC subset phenotypes analysed by flow cytometry 24 hours later.
5
Assessing HUVEC Activation by Ti3C2Tx MXene
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Activation of HUVECs in the presence and absence of Ti3C2Tx MXene nanosheets was assessed using western blot analysis for HLA class II (HLA-DRα) and ICAM-1. Briefly, HUVECs at 80% confluency were pre-treated with 2 µg/mL of Ti3C2Tx MXene for 24 h. The cells were then activated using 10 units/mL of recombinant human IFN-γ (570202, BioLegend) for 24 h and collected in RIPA buffer on ice and stored at 80 ºC for 24 h. After thawing, cell lysate was spun at 12,000 g for 10 min to collect the supernatant. Protein was quantified using the Bradford Protein Quantification Assay (5000006, Bio-Rad). For gel electrophoresis, 30 µg of protein was loaded into a 10% polyacrylamide resolving gel (1610658, Bio-Rad) and run at 100 V, and then transferred onto a PVDF membrane. The desired primary antibody was incubated with the membrane overnight at 4 °C. The secondary antibodies were added for 1 h at room temperature. Signal was detected using the Pierce™ ECL Western Blotting Substrate (32209, Thermo Fisher Scientific) and a ChemiDoc MP (Bio-Rad). Signal intensities were quantified using Quantity One 1-D Analysis Software (Bio-Rad, Hercules, California) and normalized to beta-actin. Four biological replicates were included for each treatment condition. The list of primary and secondary antibodies used for probing the membranes, along with their dilutions, is presented in Supplementary Table S10 .
6
Cisplatin Resistance in NSCLC Cells
7
Interferon-Induced Gene Expression Analysis
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HEK293, A549, and Calu-3 cells were incubated with different concentrations of recombinant human IFN-α2 (103, 104, and 105 U/mL; BioLegend), recombinant human IFN-β (103, 104, and 105 U/mL; PeproTech), recombinant human IFN-ω (105, 106, and 107 U/mL; PeproTech), recombinant human IFN-γ (103, 104, and 105 U/mL; BioLegend), and recombinant human IFN-λ2 (103, 104, and 105 U/mL; PeproTech), respectively. At 24 h poststimulation, the cells were harvested, and the gene expression levels of α-SNAP, ISG56, IP-10, and MX1 were determined by qPCR.
A549 cells were incubated with IFN-α2 (104 U/mL), IFN-β (104 U/mL), IFN-γ (104 U/mL), IFN-λ2 (104 U/mL), and IFN-ω (106 U/mL), respectively. At 12, 24, and 48 h poststimulation, the cells were harvested to determine α-SNAP mRNA levels by qPCR. The α-SNAP protein expression levels at 24 h poststimulation were determined by using flow cytometry.
A549 cells were incubated with IFN-α2 (104 U/mL), IFN-β (104 U/mL), IFN-γ (104 U/mL), IFN-λ2 (104 U/mL), and IFN-ω (106 U/mL), respectively. At 12, 24, and 48 h poststimulation, the cells were harvested to determine α-SNAP mRNA levels by qPCR. The α-SNAP protein expression levels at 24 h poststimulation were determined by using flow cytometry.
8
T Cell Activation and Tumor Cell Coculture
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T cells were sorted from Met1 and Met2 tumor digest as described above.
Met1 and Met2 tumor cells lines were derived from sorted tumor cells and cultured in RPMI 1640 (Gibco) supplemented with 10% FCS, 100 U/mL Penicillin, 100 µg/mL Streptomycin and 2 mM L-Glutamine (RPMI-10%) and were stimulated with 2 ng/mL of recombinant human IFNγ (Biolegend) for 3 days before coculture with sorted T cells.
T cells were cultured at 1:1 ratio with tumor cell line irradiated with 30 cGy in complete human media, in presence of rhIL7 and IL15 (both Peprotech, 25 ng/mL) and human IL-2 (Peprotech, 20 U/mL), the last one added only from day 3 of culture. Half of the media was changed every second day. On day 11, one-third of the cells were restimulated with one-third of the original numbers of tumor cells in the presence or absence of BD GolgiPlug (Brefeldin A). Cells were stained as described above adding anti-CD25 BV605 (Biolegend).
Met1 and Met2 tumor cells lines were derived from sorted tumor cells and cultured in RPMI 1640 (Gibco) supplemented with 10% FCS, 100 U/mL Penicillin, 100 µg/mL Streptomycin and 2 mM L-Glutamine (RPMI-10%) and were stimulated with 2 ng/mL of recombinant human IFNγ (Biolegend) for 3 days before coculture with sorted T cells.
T cells were cultured at 1:1 ratio with tumor cell line irradiated with 30 cGy in complete human media, in presence of rhIL7 and IL15 (both Peprotech, 25 ng/mL) and human IL-2 (Peprotech, 20 U/mL), the last one added only from day 3 of culture. Half of the media was changed every second day. On day 11, one-third of the cells were restimulated with one-third of the original numbers of tumor cells in the presence or absence of BD GolgiPlug (Brefeldin A). Cells were stained as described above adding anti-CD25 BV605 (Biolegend).
9
PARP Inhibitor and Immune Checkpoint Antibody Protocol
10
Quantifying Cytotoxic Activity of Natural Killer Cells
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