The following antibodies from BD Biosciences were used for flow cytometry: biotinylated antibodies to CD4 (553728), CD8 (553029), IgM (553436), CD11b (553309), CD138 (553713) and GR-1 (553125); PE-conjugated antibodies to B220 (553090), CD5 (553023), CD11b (557397), IgD (558597), CD138 (553714); APC-conjugated antibodies to CD21 (558658), IgM (550676), IgD (560868), CD11b (553312), CD138 (558626) and GR-1 (553129); FITC-conjugated antibodies to GL7 (553666), IgD (553439); PE-Cy7 conjugated anti-CD11b (552820); Pacific Blue anti-CD3e (558214); PE-Cy5-anti-CD4 (553050) and APC-Cy7-anti-CD8a (557654). From Biolegend: APC-anti-mouse IgG1 (406610), PerCPCy5.5-anti-mouse IgG1(406612), Pacific Blue anti-CD38 (102720), FITC-anti-mouse IgG (405305), APC-anti-mouse IgG (405308), APC-Cy7-anti-mouse IgG (405316). From eBioscience: PerCP-Cy5.5-anti-B220 (45-0452-82), PE-anti-IgM (12-5890-83), PE-anti-CD23 (12-0232-82), Biotin-anti-mouse-IgD (13-5993-85), Streptavidin-PE (12-4317-87), PE-Cy7-streptavidin (25-4317-82). From the Jackson Immunoresearch Laboratories: normal rabbit IgG (015-000-002) or mouse IgG (011-000-002). From Abgent: anti-LC3 (AP1802a) for immunocytochemistry. From Invitrogen: Anti-CoxIV (459600) for immunocytochemistry. From Southern Biotechnology, HRP conjugated anti-mouse IgG or IgM.
Fitc anti mouse igg
Manufactured by BioLegend
Sourced in United States
FITC anti-mouse IgG is a fluorescently labeled secondary antibody that binds to mouse immunoglobulin G (IgG). It is designed for use in various immunoassays and cellular analysis techniques to detect and visualize the presence of mouse IgG proteins.
Automatically generated - may contain errors
Lab products found in correlation
Streptavidin pe, by Thermo Fisher Scientific (1 mentions)
Streptavidin pe cy7, by Thermo Fisher Scientific (1 mentions)
Pe cy5 anti cd4, by BD (1 mentions)
Pe anti igm, by Thermo Fisher Scientific (1 mentions)
Percp cy5.5 anti b220, by Thermo Fisher Scientific (1 mentions)
Pacific blue anti cd38, by BioLegend (1 mentions)
Biotin anti mouse igd, by Thermo Fisher Scientific (1 mentions)
Normal rabbit igg, by Jackson ImmunoResearch (1 mentions)
Facsverse flow cytometer, by BD (1 mentions)
Cell staining buffer, by BD (1 mentions)
Cfse dye, by BioLegend (1 mentions)
Facscanto 2, by BD (1 mentions)
Pe anti mouse igg, by BioLegend (1 mentions)
Facscalibur, by BD (1 mentions)
B220 microbeads, by Miltenyi Biotec (1 mentions)
Lsrfortessa x 20, by BD (1 mentions)
Pd 1 pe, by BioLegend (1 mentions)
Cd11b fitc, by BioLegend (1 mentions)
Annexin 5 allophycocyanin, by BioLegend (1 mentions)
Fcs express v4, by De Novo Software (1 mentions)
11 protocols using fitc anti mouse igg
1
Comprehensive Flow Cytometry Antibody Panel
2
Porcine T Cell Proliferation Assay
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For T cell proliferation analysis, PBMCs from Tg and WT pigs were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) dye (BioLegend) as previously described (44 (link)). Briefly, cells were incubated with 5 µM CFSE in PBS containing 5% FBS at 37°C for 5 min and washed three times with ice-cold PBS-5% FBS. 2 × 105 CFSE-labeled PBMCs/well of a 96-well plate were cultured in RPMI-1640 media supplemented with 10% heat-inactivated FBS, 1% penicillin/streptomycin in the presence of stimulation. PBMCs were harvested after a 3-day culture for subsequent analysis by the BD FACSVerse™ flow cytometer.
Peripheral blood mononuclear cells were cultured in 24-well round bottom plates (1 × 106 cells/well) and stimulated for 0, 24, 48, and 72 h with ConA (2 µg/mL), recovered by centrifugation and re-suspended in 100 µL of PBS containing 10% porcine serum. After being washed twice with a cell-staining buffer (BD Biosciences), the cells were suspended in 50 µL of staining buffer and stained with mouse anti-pig 4-1BB monoclonal antibody at 4°C for 30 min. After two washes, they were stained with FITC anti-mouse IgG (BioLegend Cat. No. 406001). Then, the cells were washed two times, suspended in 200 µL of sterile PBS, and analyzed using the BD FACSVerse™ flow cytometer.
Peripheral blood mononuclear cells were cultured in 24-well round bottom plates (1 × 106 cells/well) and stimulated for 0, 24, 48, and 72 h with ConA (2 µg/mL), recovered by centrifugation and re-suspended in 100 µL of PBS containing 10% porcine serum. After being washed twice with a cell-staining buffer (BD Biosciences), the cells were suspended in 50 µL of staining buffer and stained with mouse anti-pig 4-1BB monoclonal antibody at 4°C for 30 min. After two washes, they were stained with FITC anti-mouse IgG (BioLegend Cat. No. 406001). Then, the cells were washed two times, suspended in 200 µL of sterile PBS, and analyzed using the BD FACSVerse™ flow cytometer.
3
Flow Cytometric Analysis of MSCs
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MSCs were detached with trypsin-EDTA, collected and 1.0 × 104 aliquots of cells were incubated with antibodies (antibody 1 μl/PBS 500 μl) for 15 minutes at 4°C. The cells were then washed with cold PBS and fixed with 4% paraformaldehyde. Antibodies attached to specific cell surface proteins were detected with a FACS Canto II machine (Becton Dickinson Biosciences, Bedford, MA, USA) and analyzed using FlowJo 10.4 software (FlowJo). The following antibodies were used: FITC anti-human HLA-ABC, FITC anti-mouse IgG, and PE anti-mouse IgG (all sourced from BioLegend, San Diego, CA, USA).
The remaining contents are in the supplementary methods.
The remaining contents are in the supplementary methods.
4
Quantifying Antibody Levels in Mice
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Spleen cells derived from BALB/c mice were harvested and their red blood cells were lysed. B cells in splenocytes were first depleted using B220 microbeads (Miltenyi Biotec) via negative selection. Cells (1 × 106/sample) then were stained with diluted serum (1/10) from naïve or transplanted C57BL/6 mice. They were further incubated with PE-anti-mouse IgM or FITC-anti-mouse IgG (Biolegend). The mean fluorescence intensity was determined by a flow cytometer (FACSCalibur, BD Biosciences).
5
Characterizing Immune Cells in Murine Tumor Model
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Spleens and metastatic tumor lesions of FVBN202 mice were harvested when the animals became moribund and were then separately homogenized into a single cell suspension as described previously [11 (link)] and below. Splenocytes were then characterized using flow cytometry. Reagents used for flow cytometry include the following: anti-CD16/32 antibody (clone 93), FITC-CD3 (17A2); FITC-CD11b (M1/70); FITC-anti-mouse IgG (Poly4053); PE-GR-1 (RB6-8C5); PE-PD-1 (RMP1-30); PE-CD25 (3C7); PE-Ki67 (16A8); allophycocyanin-CD49b (DX5); allophycocyanin-CD62 ligand (MEL-14); allophycocyanin-Annexin V; PerCP/CY5.5-CD4 (GK1.5); PE/CY7-CD8α (53-6.7); Brilliant Violet 421-PD-L1 (10F.9G2); Brilliant Violet 605-CD45 (30-F11); and PI, all of which were purchased from BioLegend (San Diego, CA, USA). BD Horizon V450-Annexin V and FITC-FVS were purchased from BD Biosciences. Anti-rat neu antibody (anti–c-Erb2/c-Neu; 7.16.4), was purchased from Calbiochem. All reagents were used at the manufacturer’s recommended concentration. Cellular staining was performed as described previously by our group [11 (link)] or as recommended by the manufacturer (Ki67, FVS). Multicolor data acquisition was performed using a LSRFortessa X-20 (BD Biosciences). Data were analyzed using FCS Express v4.07 (De Novo Software, Glendale, CA, USA).
6
Dot-Blot Immunoassay for Recombinant OmpA-LTB
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The dot-blot immunoassay was performed on nitrocellulose membrane (Sigma-Aldrich). Recombinant protein OmpA-LTB was spotted on the membrane by placing 1 μl of antigen solution (12mg/ml in PBS). The membrane was air-dried for 15 minutes at room temperature. Then it was blocked in 5% nonfat dried milk in TBS at 37°C for 2 hours with gentle shaking. After blocking, the dotted regions of membrane were incubated for 1 hour with mouse serum which is considered as primary antibody. After washing the membrane with TBS, 2 μl of 0.5 mg/ml FITC anti-mouse IgG (BioLegend, San Diego, USA) was added to the dotted regions of membrane and incubated for 1 hour at 37°C. The membrane was washed twice with TBS and visualized by a U.V transiluminator (Labnet, Edison, NJ, USA).
7
Immunofluorescence Analysis of Mouse Kidney and Spleen
8
Measuring Antitumor Antibodies in Mice
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To assess for the presence of antitumor antibodies in treated mice, blood was collected at days 15 and 30 for analysis as previously described.30 (link) Briefly, serum components were isolated and frozen at −80°C until ready for analysis, at which point serum was thawed and co-incubated with B78 cells for antibody labeling. Labeled cells were washed and tumor bound antibody was detected using secondary antibodies [anti-mouse IgG-FITC (405305; Biolegend), anti-mouse IgG1-PE (406607; Biolegend), anti-mouse IgG2b-PE (406708; Biolegend), anti-mouse IgG2c-FITC (NBP2-68518; Novus)] and a live dead vitality stain (DAPI).
9
Phenotyping and Transduction of CAR-T Cells
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The antibodies used to identify the phenotype of CAR-T cells including anti-Human CD3 (APC-Cy7), anti-Human CD4 (PE), anti-Human CD45RO (PE-Cy7), anti-Human CD62L (FITC), and anti-Human CD8 (FITC) antibodies, as well as corresponding mouse IgG controls and anti-CD54 (FITC), were all purchased from BioLegend. ICAM1 expression levels on the surface of human tumor cells and ICAM1-CAR transduction efficiency were evaluated via purified ICAM1-scFv-Fc and ICAM1 ECD-Fc, respectively. The transduction efficiency of control CAR-T (anti-CD19) was evaluated via Myc-tag mouse mAbs staining (Cell Signaling Technologies). Anti-mouse IgG-FITC (BioLegend) was used to label the Fc of ICAM1 ECD-Fc. Fluorescence was assessed using a BD Fortessa flow cytometer and analyzed using FlowJo 10.6.0 software.
10
Comprehensive Immunophenotyping by Flow Cytometry
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The following monoclonal antibodies were used for flow cytometry analysis and cell sorting; anti-CD8a-Alexa Fluor 488 (300916, BioLegend, 1:200), anti-CD8a-FITC (FITC-65135, Proteintech, 1:50), anti-CD3-PE (317307, BioLegend, 1:200), anti-CD4-APC (300552, BioLegend, 1:200), anti-CD45RA-FITC (304148, BioLegend, 1:200), anti-CD45RO-PerCP/Cy5.5 (304222, BioLegend, 1:200), anti-PD-1-APC/Cy7 (329921, BioLegend, 1:50), anti-CD57-PerCP/Cy5.5 (359621, BioLegend, 1:200), anti-IFNγ-APC (502511, Biolegend, 1:200), anti-TNFα-BV421 (502931, BioLegend, 1:100), anti-CCR7-PE (353203, BioLegend, 1:50), anti-TIGIT-PerCP/Cy5.5 (372717, BioLegend, 1:50), anti-pan HLA (M0736, Dako, 1:200), control mouse IgG2a (401501, BioLegend, 1:1111), and anti-mouse IgG-FITC (406001, BioLegend, 1:200). LIVE/DEAD Fixable-Near IR Dead Cell Stain Kit (L10119, Thermo Fisher Scientific, 1:400), LIVE/DEAD Fixable-Aqua Dead Cell Stain Kit (L34957, Thermo Fisher Scientific, 1:400), and Human BD Fc Block (564220, BD Pharmingen, 1:50) was also used. MHC tetramer was prepared using QuickSwitch Quant HLA-A*24:02 Tetramer Kit-PE (TB-7302-K1, MBL Life Science) and QuickSwitch Quant HLA-A*24:02 Tetramer Kit-BV421 (TB-7302-K4, MBL Life Science) with appropriate peptides. CMV pp65341-349 peptide (TS-0020-P, MBL) was purchased and all the other peptides were synthesized with a purity of >95% (Scrum Inc., or Toyobo).
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