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).
F ab 2 fragment specific antibody
Manufactured by Jackson ImmunoResearch
Sourced in United States
F(ab')2 fragment specific antibody is a laboratory reagent used to detect and analyze specific antibodies in a sample. It is a fragment of an antibody molecule that retains the antigen-binding capacity but lacks the Fc region. This reagent is commonly used in various immunoassay techniques, such as ELISA, Western blotting, and immunohistochemistry, to identify and quantify target antibodies in complex biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2, by BD (3 mentions)
Fetal bovine serum (fbs), by GE Healthcare (3 mentions)
Phosphate buffered saline (pbs), by Lonza (3 mentions)
Clone fm276, by Miltenyi Biotec (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by BD (2 mentions)
Cd4 clone sk3, by BD (2 mentions)
B7 h3 antibody clone 7 517, by BD (2 mentions)
Cd45ro clone uchl1, by BD (2 mentions)
Ccr7 clone g043h7, by BioLegend (2 mentions)
Cd8 clone sk1, by BD (2 mentions)
Streptavidin pe, by BioLegend (1 mentions)
Mouse gamma globulin, by Jackson ImmunoResearch (1 mentions)
Lag 3 rea351, by Miltenyi Biotec (1 mentions)
Biotin sp conjugated goat anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Facscanto flow cytometer, by BD (1 mentions)
Annexin 5, by BD (1 mentions)
4 protocols using f ab 2 fragment specific antibody
1
Multiparametric Immunophenotyping of CAR T Cells
2
Flow Cytometry Analysis of CAR T Cells
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A FACSCanto II (BD Biosciences) instrument was used to acquire flow cytometry data, which was analyzed using FlowJo v10.7 (BD Biosciences). For surface staining, samples were washed with and stained in PBS (Lonza) with 1% FBS (GE Healthcare). For all experiments, matched isotypes or known negatives (e.g. nontransduced T cells or B7-H3-negative cell lines) served as gating controls. CAR detection was performed using F(ab’)2 fragment-specific antibody (polyclonal, Jackson ImmunoResearch, West Grove, PA, USA). T cells were stained with fluorochrome-conjugated antibodies using combinations of the following markers: CD4 (clone SK3, BD Biosciences), CD8 (clone SK1, BD Biosciences), CCR7 (clone G043H7, BioLegend, San Diego, CA, USA), and CD45RO (clone UCHL1, BD Biosciences). LM7 and the negative control leukemia cell line BV173 were evaluated for expression of B7-H3 using B7-H3 antibody (clone 7-517, BD Biosciences, or clone FM276, Miltenyi). Cells were additionally stained with DAPI (BD Biosciences) to gate for live cells.
3
Flow Cytometric Analysis of CAR T Cells
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A FACSCanto II (BD Biosciences) instrument was used to acquire flow cytometry data, which was analyzed using FlowJo v10 (BD Biosciences). For surface staining, samples were washed with and stained in PBS (Lonza) with 1% FBS (GE Healthcare). For all experiments, matched isotypes or known negatives (e.g., NT T cells) served as gating controls. CAR detection was performed using F(ab′)2 fragment=specific antibody (polyclonal, Jackson ImmunoResearch, West Grove, PA, USA). T cells were stained with fluorochrome-conjugated antibodies using combinations of the following markers: CD4 (clone SK3, BD Biosciences), CD8 (clone SK1, BD Biosciences), CCR7 (clone G043H7, BioLegend, San Diego, CA, USA), CD45RO (clone UCHL1, BD Biosciences), and 41BBL (clone 5F4, BioLegend). Tumor cell lines were evaluated for expression of B7-H3 using B7-H3 antibody (clone 7-517, BD Biosciences, or clone FM276, Miltenyi). To determine apoptosis, T cells were labeled with annexin V (BD Biosciences) and DAPI (BD Biosciences). The percentages of apoptotic cells were determined by the percent annexin V positive.
4
Flow Cytometry Protocol for CAR and B7-H3 Expression
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A FACSCanto II (BD) instrument was used to acquire flow cytometry data, which was analyzed using FlowJo v10 (BD). For surface staining, samples were washed with and stained in PBS (Lonza) with 1% FBS (GE Healthcare Life Sciences). For all experiments, matched isotypes or known negatives (e.g., B7-H3 knockout cells) served as gating controls. CAR detection was performed using F(ab’)2 fragment specific antibody (polyclonal, Jackson ImmunoResearch, West Grove, PA). Tumor cell lines were evaluated for expression of B7-H3 using B7-H3 antibody (clone 7–517, BD or clone FM276, Miltenyi).
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