UAMC-1110 was generously provided by Dr. Pieter Van der Veken (University of Antwerp). Unless otherwise specified, UAMC-1110 was administered in high molecular weight PEG at a concentration of 20mg/kg by oral gavage twice per day. Fluorescently-conjugated antibodies CD3-e450, CD8-PerCP, CD4-FITC, CD4-e450, CD4-PerCP, CD25-APC, IFNγ-APC, IL-2-PE, TNFa-PE-Cy7, CD11b-PE-Cy7, Ly6G-FITC, Ly6C-PerCP-Cy5.5, Gr1-PE-Cy7, and MHCII-EF450, PDGFRβ-APC, CD31-PE, CD45-BV510, and EpCam-BV605 were purchased from Thermo Fisher Scientific (Lafayette, CO) or BD Biosciences (San Jose, CA). CD8-PE-TxRD was purchased from Invitrogen (Carlsbad, CA). SIY, SIINFEKL, and β-galactosidase peptides were obtained from Integrated DNA Technologies (Coralville, Iowa). β-galactosidase (βgal) for the ICPMYARV peptide and SIINFEKL peptide tetramers were obtained from the NIH Tetramer core facility (Atlanta, GA). Antibodies to CD31 (Thermo Fisher Scientific), αSMA (Sigma-Aldrich, St Louis, MO), PDGFRα (Thermo Fisher Scientific), Vimentin (Cell Signaling Technology, MA), Ki67 (Abcam), Cleaved Caspase 3 (Cell Signaling Technology) were used for immunofluorescent staining and hypoxyprobe Omni Kit (Burlington, MA) was used for hypoxia staining. Anti-PD1 antibody clone RPM1-14 (BioXcell, West Lebanon, NH) was used where indicated for treatment studies.
Apc ifnγ
Manufactured by Thermo Fisher Scientific
Sourced in United States
The APC-IFNγ is a lab equipment product that detects and measures interferon-gamma (IFNγ) in biological samples. It uses flow cytometry technology to provide quantitative analysis of IFNγ-producing cells.
Automatically generated - may contain errors
Lab products found in correlation
Ionomycin, by Merck Group (3 mentions)
Facscanto 2, by BD (3 mentions)
Foxp3 pe, by BioLegend (2 mentions)
Fixation and permeabilization buffer, by Thermo Fisher Scientific (2 mentions)
Streptavidin pe, by Thermo Fisher Scientific (2 mentions)
Cd3 efluor450 17a2, by Thermo Fisher Scientific (2 mentions)
Biotin tcrγδ, by Thermo Fisher Scientific (2 mentions)
Cd69 fitc, by Thermo Fisher Scientific (2 mentions)
Pecy7 pannk, by Thermo Fisher Scientific (2 mentions)
Fixable viability dye, by Thermo Fisher Scientific (2 mentions)
Golgiplug, by BD (2 mentions)
Ly6g fitc, by Thermo Fisher Scientific (1 mentions)
Cd11b pe cy7, by Thermo Fisher Scientific (1 mentions)
Gr 1 pecy7, by Thermo Fisher Scientific (1 mentions)
Cd4 fitc, by Thermo Fisher Scientific (1 mentions)
Cd4 percp, by Thermo Fisher Scientific (1 mentions)
Cd25 apc, by Thermo Fisher Scientific (1 mentions)
Ly6c percp cy5, by Thermo Fisher Scientific (1 mentions)
Cd8 pe txrd, by Thermo Fisher Scientific (1 mentions)
Pdgfrα, by Thermo Fisher Scientific (1 mentions)
7 protocols using apc ifnγ
1
UAMC-1110 Immunotherapy Protocol
2
Intracellular Cytokine Detection in CD4+ T Cells
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For the detection of intracellular cytokines, cells were reactivated by PMA (50 ng/ml) and ionomycin (500 ng/ml) (Sigma‐Aldrich) with protein transport inhibitor (Thermo Fisher Scientific, Waltham, MA, USA) for 5 h. Cells were blocked by anti‐mouse CD16/32 antibodies before surface staining. Surface proteins expressed on cells were stained with fluorescence‐conjugated antibodies diluted in FACS buffer (1× phosphate‐buffered saline with 0.5% bovine serum albumin) for 30 min. Intracellular cytokine staining was performed using intracellular fixation and permeabilization buffer set (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer's recommended protocol. The anti‐mouse CD4‐PerCP‐Cy7, IL‐17A‐APC, IL‐17A‐PE, IFNγ‐APC and IFNγ‐PE fluorescence‐conjugated antibodies for flow cytometry were purchased from Thermo Fisher Scientific (Waltham, MA, USA). After staining, cells were washed and acquired on FACS Canto II (BD Biosciences, Franklin Lakes, NJ, USA). To figure out cytokine‐producing CD4 T cells, lymphocytes were gated from FSC‐A and SSC‐A dot plot. Next, single cells were gated from FSC‐H and FSC‐A. CD4+ cells were gated from CD4‐Percp‐cy5.5 and FSC‐A dot plot. And then, intracellular cytokines in CD4 T cells were measured. Data were analysed by using FlowJo software (FlowJo, LLC, Ashland, OR, USA).
3
Multiparametric Flow Cytometry Analysis
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PE-Cy™7-CD4 (Clone: RM4-5), APC-Cy™7-CD69 (Clone: H1.2F3), APC-Cy™7-CD45 (Clone: 30-F11), APC-Cy™7-CD19 (Clone: 6D5), PE-Cy™7-MHC II (Clone: M5/114.15.2), PE-Foxp3 (Clone: MF-14), FITC-Ly6C (Clone: HK1.4), BV421-CD11b (Clone: M1/70) were purchased from Biolegend. PE-F4/80 (Clone: BM8), APC-IFN-γ (Clone: XMG1.2), Biotin-CD11c (Clone: N418) were purchased from eBioscience. APC-Cy™7-CD4 (Clone: GK1.5), APC-CD40 (Clone: HM40-3), APC-CD25 (Clone PC61), PerCP-Cy™5.5-CD80 (Clone: 16-10A1), FITC-CD3e (Clone: 145-2C11), PE-IL-17a (Clone: TC11-18H10), Sav-BUV395 were purchased from BD Biosciences. Flow cytometry data were acquired on BD LSR II flow cytometer as previously described40 and analyzed with FlowJo™ v10.7.
4
Molecular Analysis of Cell Signaling Pathways
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Dulbecco’s Modified Eagle Medium (DMEM/HEPES, Cat #12430-054) and Penicillin/Streptomycin (Cat #15240062) were from Gibco (Carlsbad, CA). Fetal bovine serum (FBS) and G418 sulfate were obtained from Invitrogen (Carlsbad, CA). Rabbit antibody against β-actin (Cat #4970S) was from Cell Signaling (Beverly, MA). Rabbit antibody against NHE1 (Cat #ab67314) and rat antibody against CD8 (Cat #ab22378) were from Abcam Ltd. (Cambridge, MA). Rat anti-mouse CD31 (Cat #550274) was from BD Pharmingen (San Jose, CA). Rabbit anti-ionized calcium-binding adapter molecule 1 (iba1) was from Wako (Richmond, VA). PerCP/Cy5.5-CD45, PE-P2RY12, BV421-TGFβ, BV605-TNFα, APC/780-IL-1β, PE/Cy7-IL10, PerCP/Cy5.5-CD8a, APC/Cy7-CD4, Alexa Fluor 700-CD25, PE-FoxP3, BV605-PD-1, PE-Gr-1, APC-NK1.1, and Pacific Blue Granzyme B were obtained from Biolegend (San Diego, CA). eFluor 450-CD16/32, PE/Cy7-CD206, APC-IFNγ was purchased from eBioscience (San Diego, CA). APC-IL-6 were obtained from thermos fisher (Waltham, MA). BUV737-CD11b, Alexa Flour 700-CD86 were obtained from BD Biosciences (San Jose, CA). PE-Ym1were from Abcam Ltd. (Cambridge, MA). Rabbit anti-Laminin (Cat #L9393) and cariporide (HOE642) was purchased from Sigma Chemicals (St. Louis, MO). Anti-mouse PD-1 (RMP1-14) and IgG2a isotype control were from BioXcell (West Lebanon, NH).
5
Lung Single-Cell Immunophenotyping Protocol
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Lung single cell suspensions were prepared using a standardized protocol as previously described38 (link). Cells were stained with the following antibodies from BD Biosciences and eBioscience: eFluor450-CD3 (17A2), PerCP-CD4 (RM4-5), FITC-CD8a (53-6.7), Biotin-TCRγδ (GL3), PE-Streptavidin, APC-IL-17A (eBio17B7), APC-IFNγ (XMG1.2), FITC-CD69 (H1.2F3), PerCP-Cy5.5-Gr1 (RB6-8C5), and PECy7-panNK (DX5). For intracellular staining, cells were stimulated for 5 h with 5 ng/ml PMA and 500 ng/ml ionomycin (Sigma-Aldrich; St. Louis, MO, USA) in the presence of a protein-transport inhibitor (GolgiPlug, BD Biosciences; San Jose, CA, USA). Following stimulation, cells were collected, stained with a fixable viability dye (eBioscience), fixed, permeabilized (Fixation and Permeabilization Buffer; eBioscience), then intracellularly stained. Samples were read using a FACSCanto II (BD Biosciences) flow cytometer and analyzed with FlowJo software (version 7.6.5 for Windows; Tree Star; Ashland, OR, USA). To simultaneously compare both quantity (% of cells) and quality (intensity of staining) of IL-17-producing populations, integrated median fluorescent intensity (iMFI) was calculated by multiplying percent of x cell type (i.e., CD4, CD8, γδ, Gr1, NK) of total IL-17A+ cells multiplied by the IL-17A MFI of x cell type.
6
Flow Cytometry Analysis of Cell Surface Markers
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Flow cytometry was used for the analysis of cell surface markers [41 (link)]. Cells were resuspended using PBS, and then were labeled with antibodies including PE anti-mouse MHCII (11-5321-81), FITC anti-mouse CD11b(RM2801), FITC anti-mouse Sca-1(11-5981-82), FITC anti-mouse CD34(11-0349-42), FITC anti-mouse CD45(11-0451-82), PE anti-mouse CD31(12-0311-82), PE anti-mouse CD29(12-0291-82), PE anti-mouse CD90(12-0909-42), or PE rat Ig2a isotype control (12-4301-81), or FITC rat Ig2a isotype control (PA5-33193) were purchased eBioscience. Subsequently, the cells were incubated in dark for 30 min, followed by washing with PBS. Cells were examined by flow cytometry with a FACS Calibur system [42 (link)].
For intracellular staining, splenic lymphocytes were collected from mice and stimulated for 4–6 h with Cell Stimulation Cocktail (eBioscience, 00-4970-03c), then the cells were stained with FITC-CD3 (eBioscience,11-0032-80) and PE-CD4 (eBioscience, MCD0404) for 30 min at 4 °C. Cells were then fixed and permeabilized using the Fixation/Permeabilization Diluent (eBioscience, 00-5223-56), according to the manufacturer’s instructions. For cytokine staining, the cells were stained with APC-IFN-γ (eBioscience, 17-7311-82) and measured with flow cytometry [43 (link)]. Data were analyzed with FlowJo.
For intracellular staining, splenic lymphocytes were collected from mice and stimulated for 4–6 h with Cell Stimulation Cocktail (eBioscience, 00-4970-03c), then the cells were stained with FITC-CD3 (eBioscience,11-0032-80) and PE-CD4 (eBioscience, MCD0404) for 30 min at 4 °C. Cells were then fixed and permeabilized using the Fixation/Permeabilization Diluent (eBioscience, 00-5223-56), according to the manufacturer’s instructions. For cytokine staining, the cells were stained with APC-IFN-γ (eBioscience, 17-7311-82) and measured with flow cytometry [43 (link)]. Data were analyzed with FlowJo.
7
Lung Single-Cell Immunophenotyping Protocol
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