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.
Clone fm276
Manufactured by Miltenyi Biotec
The Clone FM276 is a laboratory instrument designed for the analysis and sorting of cells. It utilizes flow cytometry technology to detect and isolate specific cell populations based on their physical and fluorescent characteristics.
Automatically generated - may contain errors
Lab products found in correlation
F ab 2 fragment specific antibody, by Jackson ImmunoResearch (3 mentions)
Fetal bovine serum (fbs), by GE Healthcare (3 mentions)
Facscanto 2, by BD (3 mentions)
Phosphate buffered saline (pbs), by Lonza (3 mentions)
Cd4 clone sk3, by BD (2 mentions)
Cd8 clone sk1, by BD (2 mentions)
B7 h3 antibody clone 7 517, by BD (2 mentions)
Ccr7 clone g043h7, by BioLegend (2 mentions)
Cd45ro clone uchl1, by BD (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by BD (2 mentions)
Annexin 5, by BD (1 mentions)
3 protocols using clone fm276
1
Flow Cytometry Analysis of CAR T Cells
2
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.
3
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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