Cd19 allophycocyanin

Manufactured by BD

CD19-allophycocyanin is a fluorescent-labeled antibody used for the detection and identification of CD19-positive cells in flow cytometry applications. It binds specifically to the CD19 antigen expressed on the surface of B cells.

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CD19-allophycocyanin (HIB19; (3 citations) CD19-allophycocyanin (APC (2 citations)

5 protocols using cd19 allophycocyanin

Multicolor Flow Cytometry Analysis

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The following mAbs were used: CD14–peridinin-chlorophyll-protein, CD19-allophycocyanin (BD Biosciences, San Jose, Calif), CD1c-allophycocyanin, CD141-allophycocyanin, and CD303-phycoerythrin (Miltenyi Biotec). Isotype controls were used to detect nonspecific binding. Flow cytometry was performed with a FACSCanto II (BD Biosciences) and analyzed with FACSDiva software (BD Biosciences) and FlowJo software (TreeStar, Ashland, Ore).

Kitzmüller C., Zulehner N., Roulias A., Briza P., Ferreira F., Faé I., Fischer G.F, & Bohle B. (2015). Correlation of sensitizing capacity and T-cell recognition within the Bet v 1 family. The Journal of Allergy and Clinical Immunology, 136(1), 151-158.

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T-B Cell Activation and Phenotyping

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Day 3 primary T cells and day 1 primary B cells were incubated with peptide and anti-CD40 as described above in 1:1 and 1:10 T/B cell ratios at a total density of 5 × 10⁶ cells per/mL. Three days later they were stained with anti CD4 allophycocyanin (ebioscience clone GK1.5), ICOS PE (ebioscience clone 7E.17G9) and PD1 FITC (ebioscience clone J43) for T-cell marker analysis. CXCR5 bio (BD clone 2G8) was used in combination with streptavidin PerCP (BD). For B-cell staining, GL7 FITC (BD), CD95 PE (BD) and CD19 allophycocyanin (BD, clone 1D3) were used.

Sinai P., Dozmorov I.M., Song R., Schwartzberg P.L., Wakeland E.K, & Wülfing C. (2014). T/B cell interactions are more transient in response to weak stimuli in SLE-prone mice. European journal of immunology, 44(12), 3522-3531.

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Flow Cytometric Crossmatch Assay

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Flow cytometric crossmatches (FCXMs) were performed with selected eluates prepared from 10–day stimulated B–cell cultures using standard FCXM protocols. Briefly, HLA typed PBMC (0.5 × 10⁶) from healthy individuals were incubated with 25 μL of IgG isolated supernatants or specific control sera for 30 minutes at room temperature. After washing three times with phosphate–buffered saline, samples were stained with mouse antihuman CD3–phycoerythrin (clone:SK7), CD19–allophycocyanin (clone: HIB19, both from BD Biosciences, Breda, the Netherlands) and rabbit anti–human IgG F(ab´)₂–fluorescein isothiocyanate (Dako, Leiden, the Netherlands) for 30 minutes, in the dark at 4°C. After 2 wash steps with phosphate–buffered saline, cells were fixed in 1% paraformaldehyde and acquired on an Accuri C6 flow cytometer (BD Biosciences). T–cell crossmatches were considered positive if the ratio of the MFI of the sample to the MFI of the negative control serum was greater than 1.6. For B–cell crossmatches, an MFI ratio above 2.6 was considered positive.

Karahan G.E., Krop J., Wehmeier C., de Vaal Y.J., Langerak–Langerak J., Roelen D.L., Lardy N.M., Bemelman F.J., ten Berge I.J., Reinders M.E., van Kooten C., Claas F.H, & Heidt S. (2019). An Easy and Sensitive Method to Profile the Antibody Specificities of HLA–specific Memory B Cells. Transplantation, 103(4), 716-723.

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Fluorescence-Activated Cell Sorting for Leukemia Subtypes

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The following monoclonal antibodies were used: mouse anti–human CD34-fluourescein isothyocyanate, CD19-allophycocyanin, CD5–brilliant violet 421, and CD38 APC-cyanine 7 (all antibodies from BD PharMingen, San Diego, CA). For some experiments, CD34-brilliant violet 421 was used, and the results were the same. After the addition of 2 μg/mL propidium iodide (BD PharMingen) to discriminate dead cells, cells were analyzed and/or sorted with a FACSAria (BD Biosciences, San Jose, CA) or a MoFlo Legacy (Beckman Coulter, Indianapolis, IN). CD34⁺CD19⁺, CD34⁻CD19⁺, and CD34⁺CD19⁻ fractions were sorted onto slides for FISH analysis and into tubes for purity checks. Following sorting, the fractions were found to be ≥96% pure.

Perkins B., Showel M., Schoch L., Imus P.H., Karantanos T., Yonescu R., Morsberger L., Ghiaur G., Gladstone D.E, & Jones R.J. (2022). CD34+ cell of origin for immunoglobulin heavy chain variable region unmutated, but not mutated, chronic lymphocytic leukemia. Leukemia & lymphoma, 63(7), 1617-1623.

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Macrophage-Mediated Phagocytosis of CLL Cells

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Monocyte-derived macrophages were generated as described [36] . Chronic lymphocytic leukemia target cells were stained with 0.1 mM carboxyfluorescein succinimidyl ester (Thermo Fisher Scientific) and incubated with the macrophages in the presence or absence of bsAb or control mAbs. After 2 h of incubation at 37°C, cells were harvested and stained with CD19-allophycocyanin and CD11b-phycoerythrin (BD Biosciences) and analyzed by flow cytometry. Phagocytosis was defined as the percentage of CD11b + cells that were carboxyfluorescein succinimidyl ester-positive and CD19 À [37] .

Interdonato A., Choblet S., Sana M., Valgardsdottir R., Cribioli S., Alzani R., Roth M., Duonor-Cerutti M, & Golay J. (2022). BL-01, an Fc-bearing, tetravalent CD20 × CD5 bispecific antibody, redirects multiple immune cells to kill tumors in vitro and in vivo. Cytotherapy, 24(2).

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