Immunophenotyping was performed using monoclonal antibodies directed against the following surface antigens: CD19 (HIB19 and SJ25-C1 clones), CD5 (UCHT2), CD3 (UCHT1), CD4 (RPA-T4), CD8 (SK1), CD45RA (HI100), CCR7 (150503), PD-1 (MIH4), PDL-1 (MIH1), PDL-2 (MIH18) (all from BD Biosciences), LAG-3 (REA351) and TIM-3 (F38–2E2) (Miltenyi Biotec). CD19 CAR+ T cells were detected using Biotin-SP-conjugated goat anti-mouse IgG, F (ab’)2 fragment specific antibody (#115–065-072), Mouse Gamma Globulin (#015–000-002) (both obtained from Jackson Immunoresearch), and Streptavidin-PE (Biolegend). Cells were analyzed using a FACSCanto flow cytometer (BD Biosciences), data analysis was performed using the FlowJo software (FlowJo, LLC) for the determination of frequencies and median fluorescence intensity (MFI).
Mouse gamma globulin
Manufactured by Jackson ImmunoResearch
Sourced in United States
Mouse gamma globulin is a purified protein fraction containing immunoglobulins from mouse serum. It is a commonly used reagent in immunological research and assays.
Automatically generated - may contain errors
Lab products found in correlation
Acetone, by Avantor (2 mentions)
Streptavidin alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Anti mouse ig alexa 594, by Thermo Fisher Scientific (2 mentions)
Biotinylated anti cd1a clone hi149, by BioLegend (2 mentions)
Biotinylated anti hla dr clone l243, by BioLegend (2 mentions)
Fixation perm kit, by BD (2 mentions)
Anti mouse ig apc, by BioLegend (2 mentions)
Anti cd1a fitc clone hi149, by BD (2 mentions)
Anti cd163 percp cy5.5 clone ghi 61, by BioLegend (2 mentions)
Pe anti cd14 clone hcd14, by BioLegend (2 mentions)
Anti hla dr pe cy7 clone l243, by BioLegend (2 mentions)
Facscalibur, by BD (2 mentions)
Canto 2, by BD (2 mentions)
Lag 3 rea351, by Miltenyi Biotec (1 mentions)
Biotin sp conjugated goat anti mouse igg, by Jackson ImmunoResearch (1 mentions)
F ab 2 fragment specific antibody, by Jackson ImmunoResearch (1 mentions)
Streptavidin pe, by BioLegend (1 mentions)
Facscanto flow cytometer, by BD (1 mentions)
Streptavidin pe, by BD (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
9 protocols using mouse gamma globulin
1
Multiparametric Immunophenotyping of CAR T Cells
2
Staining and visualization of skin DCs
3
Characterization of Engineered T Cells
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Transduced and non-transduced T cells were harvested and washed with FACS buffer (PBS + 5% FCS and 10% sodium azide). An aliquot was used to assay for cell viability with the trypan blue dye exclusion assay. Staining of the CD19 CAR construct was performed using a 1:5 dilution of Affinpure goat-anti mouse IgG (Jackson ImmunoResearch, West Grove, PA, USA, Cat# 115-065-072) diluted in FACS buffer. Cells were incubated for 30 min at 4 °C in this mixture, washed 2× with FACS buffer, and blocked for 20 min at 4 °C using a 1:10 dilution of mouse gamma globulin (Jackson ImmunoResearch, Cat# 015-000-002). Excess blocking was washed with FACS buffer, and cells were stained with PE streptavidin (BD Biosciences, San Jose, CA, USA, Cat# 349023) (20 min, 4 °C). Cells were washed 2× with FACS buffer and resuspended in the same buffer containing propidium iodide. Cells were subsequently analyzed by flow cytometry using an LSRII flow cytometer, BD (Franklin Lakes, NJ, USA).
4
Quantifying Skin Dendritic Cells
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Frozen skin sections were prepared from 24 h cultured skin biopsies from the ‘bath’ and injection approach and fixed in acetone (VWR, Darmstadt, Germany) for 10 min at room temperature. The targeting mAbs against Langerin and DEC-205 were visualized with anti-mouse-Ig-Alexa 594 (Invitrogen) followed by blocking residual free anti-mouse binding sites with 100 μg/ml mouse gamma globulin (Jackson ImmunoResearch Laboratories, Pennsylvania, USA) for 15 min. Skin DC were identified with biotinylated anti-CD1a (clone HI149, BioLegend) and biotinylated anti-HLA-DR (clone L243, BioLegend), followed by Streptavidin-Alexa Fluor 488 (Invitrogen). Appropriate isotype controls were purchased from Biolegend. All staining steps were performed for 30 min at 37°C.
5
Skin DC Subsets Identification
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Emigrated skin DC were collected 4 days after the start of skin explant cultures. Cells were permeabilized and stained with the BD-Fixation/Perm kit (BD Biosciences, San Diego, CA, USA) according to the manufacturer's instruction. Targeting mAbs against Langerin and DEC-205 were detected with anti-mouse-Ig-APC (BioLegend), followed by blocking with 100 μg/ml mouse gamma globulin for 5 min (Jackson ImmunoResearch Laboratories) and staining of DC with anti-CD1a-FITC (clone HI149, BD Biosciences), anti-CD163-PerCP-Cy5.5 (clone GHI/61, BioLegend), anti-CD14-PE (clone HCD14, BioLegend), anti-HLA-DR-PE-Cy7 (clone L243, BioLegend) for 10 min at 4°C. Appropriate isotype controls from Biolegend were used for FACS stainings to confirm specific staining. Analyses were performed on a FACS Calibur and Canto II instrument (BD Biosciences). The percentage of targeted skin DC was determined by pregating on viable HLA-DR+ cells, followed by gating for the various skin DC subsets with CD1a and CD14. In the three different skin DC subsets, cells positive for the targeting mAb were determined by setting the gate on DC from skin explants injected with isotype controls.
6
Comprehensive Flow Cytometry Immunophenotyping
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Flow cytometry was performed on a Beckman Coulter's Gallios. For detection of the CD19 CAR receptor we used a biotinylated anti-mouse FAB as a primary antibody (Jackson), Mouse Gamma Globulin as a blocking reagent (Jackson) and Anti-Biotin-Viogreen as a secondary antibody (Miltenyi). The following antibodies were used for additional staining: Anti Human CD3 –FITC, Anti Human TCRa/b-APC, Anti Human TCRg/d-PE, Anti Human CD19-PE, Anti Human CD10-PE-Cy7, Anti Human CD45 viogreen, Anti mouse CD45 APC, and for dead cell exclusion Ghost Red 780 Viability Dye (all from Miltenyi). All antibodies were incubated with samples for 20 min at 4°C. Analysis was done using the FlowJo analysis software V10.
7
Characterization of Skin Dendritic Cell Subsets
8
Immunoprecipitation of Golgi Proteins
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IAA-or control-treated RC65 cells were lysed in lysis buffer (50 mM Tris HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP40, protease inhibitor cocktail Complete (Roche)) for 30 min on ice.
The cell lysates were then cleared by centrifugation for 10 min at 16,000 g at 4°C. For GM130 IP, lysates were mixed with 5 µl rabbit polyclonal anti-GM130 serum or 5 µl pre-immune serum for 60 min at 4°C and incubated with protein A-Sepharose (GE Healthcare) for additional 60 min. For GRASP55 IP, cleared lysates were mixed with 2 µg mouse monoclonal anti-GRASP55 IgG or 2 µg mouse gamma globulin (Jackson ImmunoResearch) for 60 min at 4°C and incubated with protein A/G-Sepharose (Santa Cruz Biotechnology) for additional 60 min. Beads were then washed with lysis buffer and bound proteins release by boiling in SDS sample buffer.
The cell lysates were then cleared by centrifugation for 10 min at 16,000 g at 4°C. For GM130 IP, lysates were mixed with 5 µl rabbit polyclonal anti-GM130 serum or 5 µl pre-immune serum for 60 min at 4°C and incubated with protein A-Sepharose (GE Healthcare) for additional 60 min. For GRASP55 IP, cleared lysates were mixed with 2 µg mouse monoclonal anti-GRASP55 IgG or 2 µg mouse gamma globulin (Jackson ImmunoResearch) for 60 min at 4°C and incubated with protein A/G-Sepharose (Santa Cruz Biotechnology) for additional 60 min. Beads were then washed with lysis buffer and bound proteins release by boiling in SDS sample buffer.
9
CAR T Cell Surface Staining Protocol
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For CAR staining; cells were stained with a primary biotinylated anti-human Fab (for MSLN-CARs) or anti-mouse Fab (CD19-CARs) antibody (Jackson ImmunoResearch Lab) then blocked with mouse gamma globulin (Jackson ImmunoResearch Lab). Alternatively, to assess CAR transduction efficiency, MSLN CAR T cells were stained with a primary biotinylated anti-EGFRt/cetuximab antibody (R&D Systems). Cells were washed twice prior to staining with extracellular antibodies. The antibody panels used for surface staining of tumor cells can be found in Supplementary Material. Intracellular cytokine staining was performed as described previously (16) . Cells were acquired on a CytoFlex (Beckman Coulter) or FACSCanto (Becton Dickinson) flow cytometer and data were analyzed using FlowJo software (FlowJo).
Details on cytotoxicity assays, IHC quantification of tumor microarrays (TMA), measurement of soluble MSLN-related peptides (SMRP), and cytokines can be found in Supplementary Material.
Details on cytotoxicity assays, IHC quantification of tumor microarrays (TMA), measurement of soluble MSLN-related peptides (SMRP), and cytokines can be found in Supplementary Material.
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