Cd27 apc

Manufactured by Thermo Fisher Scientific

Sourced in United States

CD27-APC is a fluorescent-conjugated antibody that binds to the CD27 receptor expressed on the surface of certain immune cells. It can be used in flow cytometry analysis to identify and quantify CD27-positive cell populations.

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Close spelling variants of this product

CD27 APC-eFluor780 (O323, Anti-human-CD27-APC (clone O323), Anti-CD27 APC CD27-APC-eFluor780 CD27-APC/Cy7 APC-conjugated anti-CD27 Anti-CD27-APC-eFluor780 CD27 APC-ef780 CD27−APC and CD24-APC eFluor780

18 protocols using cd27 apc

Isolation and Analysis of CD19+CD24hiCD27+ B Cells

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PB mononuclear cells (PBMCs) and SF mononuclear cells (SFMCs) were isolated by density gradient centrifugation. To detect the proportion of CD19⁺CD24^hiCD27⁺ B cells and the expression of RANKL in CD19⁺CD24^hiCD27⁺ B cells of PBMCs or SFMCs, the cells were isolated by density gradient centrifugation and then were stained with mouse monoclonal antibodies as follow: CD19-APC/Cy7 (BioLegend, San Diego, CA, USA), CD24-FITC (eBioscience, San Diego, CA, USA), CD27-APC (eBioscience), RANKL-PE (BioLegend). FMO controls were included. Data was acquired on a FACS Arial II flow cytometer (Becton Dickinson, NJ, USA) and analysed using FlowJo software.
To isolate CD19⁺CD24^hiCD27⁺ B cells, PBMCs were stained with mouse anti-CD19-APC/Cy7, CD24-PE (eBioscience), CD27-APC, then the aimed cell populations were sorted by flow cytometry. Sorted CD19⁺CD24^hiCD27⁺ B cells had a purity of > 95%. These sorted cells were subsequently subjected to reverse transcription-polymerase chain reaction (RT-PCR) or osteoclast differentiation assay.

Guo X., Xu T., Zheng J., Cui X., Li M., Wang K., Su M., Zhang H., Zheng K., Sun C., Song S, & Liu H. (2020). Accumulation of synovial fluid CD19+CD24hiCD27+ B cells was associated with bone destruction in rheumatoid arthritis. Scientific Reports, 10, 14386.

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CAR T Cell Immunophenotyping and Transduction

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Immunophenotyping of the apheresis was conducted with the following fluorophore-conjugated antibodies: CD3-phycoerythrin (PE) (Beckman Coulter), CD8-PE-Cy7 (Invitrogen), CD14-allophycocyanin (APC) (eBioscience), and CD45-fluorescein isothiocyanate (FITC) (Beckman Coulter). In-process transduction efficiency of the CD19-targeted CAR T cells was evaluated with the CD3-APC (Invitrogen) and biotinylated goat-anti-mouse Fab (Jackson Immunoresearch Lab) followed by PE-conjugated streptavidin (MP Biomedicals). In-process transduction efficiency of the BCMA-targeted CAR T cells was evaluated with the CD3-APC (Invitrogen) and BCMA-Fc-APC.¹² (link) The effector memory and central memory immunophenotyping was conducted using the following monoclonal antibodies: CD27-APC, CD28-FITC, CD62L-FITC, CCR7-FITC, CD45RA-APC (Invitrogen), and CD127-eFlour450 (eBioscience). Dead cells were excluded from analysis using either 7AAD or DAPI staining. Flow data acquisition was performed on an LSRII (BD Biosciences), and data analysis was performed using FlowJo Software (Tree Star).

Wang X., Borquez-Ojeda O., Stefanski J., Du F., Qu J., Chaudhari J., Thummar K., Zhu M., Shen L.B., Hall M., Gautam P., Wang Y., Sénéchal B., Sikder D., Adusumilli P.S., Brentjens R.J., Curran K., Geyer M.B., Mailankhody S., O’Cearbhaill R., Park J.H., Sauter C., Slovin S., Smith E.L, & Rivière I. (2021). Depletion of high-content CD14+ cells from apheresis products is critical for successful transduction and expansion of CAR T cells during large-scale cGMP manufacturing. Molecular Therapy. Methods & Clinical Development, 22, 377-387.

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Analyzing PD-1 and PD-L1 Expression in RA

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PD-1 expression on CD4 T cells and PD-L1 expression on CD1c mDCs of RA patients were analysed by flow cytometry using a FACSCanto II flow cytometer (BD Biosciences, San Jose, CA, USA). Ex vivo or cultured mDCs were stained with CD1c phycoerythrin (CD1c-PE; BD Biosciences), CD19 peridinin chlorophyll (CD19-PerCP; BioLegend) and CD274 (PD-L1)-APC (BioLegend). mDCs were gated as CD1c-positive and CD19-negative. Ex vivo CD4 T cells were stained with CD45RO fluorescein isothiocyanate (CD45RO-FITC; Dako, Glostrup, Denmark), CD27-APC (Invitrogen), CD279 (PD-1)-PE and CD4-PerCP (BioLegend) using isotype antibodies or autofluorescence as controls. All samples were analysed using FlowJo software (TreeStar, Ashland, OR, USA). To compare mean fluorescence intensity (MFI) values, the autofluorescence intensity was subtracted from the MFI of the stains to reveal true expression values.

Moret F.M., van der Wurff-Jacobs K.M., Bijlsma J.W., Lafeber F.P, & van Roon J.A. (2014). Synovial T cell hyporesponsiveness to myeloid dendritic cells is reversed by preventing PD-1/PD-L1 interactions. Arthritis Research & Therapy, 16(6), 497.

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Single-cell sorting of plasmablasts

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PBMCs were freshly isolated from blood samples collected 7 days after PfSPZ immunization and stained for viability with Aqua Live/Dead dye(Invitrogen) followed by surface-staining for the following markers: CD20-Cy7APC (BD Bioscience), CD19-FITC (BD Bioscience), CD3-Cy7PE (BD Bioscience), CD38-PE (BD Bioscience), and CD27-APC (ThermoFisher). Plasmablasts were gated as live, CD3-CD20-CD19+CD27+CD38+ and were sorted as single cells into 96-well PCR plates containing 20 μl/well of RT reaction buffer that included 5 μl of 5× First strand cDNA buffer, 0.5 μl of RNAseOut (Invitrogen), 1.25 μl of DTT, 0.0625 μl of Igepal and 13.25 μl of dH2O (Invitrogen) as previously described^{46 (link)}.

Kisalu N.K., Idris A.H., Weidle C., Flores-Garcia Y., Flynn B.J., Sack B.K., Murphy S., Schön A., Freire E., Francica J.R., Miller A.B., Gregory J., March S., Liao H.X., Haynes B.F., Wiehe K., Trama A.M., Saunders K.O., Gladden M.A., Monroe A., Bonsignori M., Kanekiyo M., Wheatley A.K., McDermott A.B., Farney S.K., Chuang G.Y., Zhang B., Natasha K.C., Chakravarty S., Kwong P.D., Sinnis P., Bhatia S.N., Kappe S.H., Sim B.K., Hoffman S.L., Zavala F., Pancera M, & Seder R.A. (2018). A human monoclonal antibody prevents malaria infection and defines a new site of vulnerability on Plasmodium falciparum circumsporozoite protein. Nature medicine, 24(4), 408-416.

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Plasmablast Isolation from PBMCs

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Plasmablasts were isolated from freshly isolated peripheral blood mononuclear cells (PBMCs) collected seven days after Sanaria PfSPZ Vaccine immunization as previously described (Kisalu et al., 2018 (link)). Briefly, PBMCs were stained for viability with Aqua LIVE/DEAD (Thermo Fisher Scientific) followed by surface staining of the following markers: CD3-PE/Cy7 (BD Bioscience), CD19-FITC (BD Bioscience), CD20-APC/Cy7 (BD Bioscience), CD27-APC (Thermo Fisher Scientific), and CD38-PE (BD Bioscience). Plasmablasts were gated as live CD3⁻CD20⁻CD19⁺CD27⁺CD38⁺ and single cell sorted using a BD FACS Aria II (BD Immunocytometry Systems) into 96-well PCR plates containing 20 μL/well of RT reaction buffer from the SuperScript^™ First-Strand Synthesis System for RT-PCR (Thermo Fisher Scientific). Plates were snap frozen on dry ice and stored at −80°C.

Wang L.T., Hurlburt N.K., Schön A., Flynn B.J., Flores-Garcia Y., Pereira L.S., Kiyuka P.K., Dillon M., Bonilla B., Zavala F., Idris A.H., Francica J.R., Pancera M, & Seder R.A. (2022). The light chain of the L9 antibody is critical for binding circumsporozoite protein minor repeats and preventing malaria. Cell reports, 38(7), 110367.

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Flow Cytometry Analysis of Immune Cells

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The following fluorochrome-conjugated anti-human antibodies and fluorescent dyes were used for flow cytometry analyses: CD19-BV605, CD3-BV421, and CD23-BV421 (BD Biosciences, Franklin Lakes, NJ, USA); IgM-APC (Biolegend, San Diego, CA, USA); and CD19-eVolve 605, CD38-PE-eFluor 610, CD24-APC-eFluor 780, CD27-APC, CD21-PerCP-eFluor710, IgD-PE, CD5-APC, 7AAD, Fixable Viability Dye eFluor 506, Propidium Iodide, Rhodamine 123 (R123) (Thermo Fisher Scientific). Resiquimod (R848) and glucopyranosyl lipid A (GLA) were obtained from the Infectious Disease Research Institute (Seattle, WA). C12-iE-DAP (iE-DAP), polyinosinic-polycytidylic (Poly I:C), trehalose-6,6-dibehenate (TDB), and CpG ODN 2006 (CpG) were purchased from InvivoGen (San Diego, CA).

McMillan J.K., O’Donnell P, & Chang S.P. (2022). Pattern recognition receptor ligand-induced differentiation of human transitional B cells. PLoS ONE, 17(8), e0273810.

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Single-cell sorting of plasmablasts

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Phenotyping and Sorting Plasmablasts

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PBMCs obtained from the two patients were stained with titrated amounts of CD19-FITC (BD; clone HIB19), CD3-Pacific Blue (BD; clone SP34-2), CD20-PECy7 (BD; clone L27), CD27-APC (eBiosciences; clone O323) and CD38-PE (BD; clone HIT2). The plasmablast population was defined as CD3− CD19+ CD20−/low CD27+ CD38+ lymphocytes and its frequency analyzed using FlowJo software. The single-cell sorting of plasmablasts was carried out using the FACSAriaII sorter (Becton Dickinson; Franklin Lakes, NJ, USA) at the Emory University Pediatrics Flow Cytometry Core Facility under negative air pressure. The cells were sorted into a 96-well PCR plate as described in [34 (link),35 (link)], rapidly frozen on dry ice and stored at −80 °C for subsequent cDNA synthesis.

Bhaumik S.K., Priyamvada L., Kauffman R.C., Lai L., Natrajan M.S., Cho A., Rouphael N., Suthar M.S., Mulligan M.J, & Wrammert J. (2018). Pre-Existing Dengue Immunity Drives a DENV-Biased Plasmablast Response in ZIKV-Infected Patient. Viruses, 11(1), 19.

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Multicolor Flow Cytometry for T and B Cell Subsets

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PBMC were isolated from peripheral blood by Ficoll gradient and frozen for batched analysis. We designed six multicolor flow cytometry panels to quantify 60 T cell subsets along with two B cell subsets, and calculated the CD4⁺/CD8⁺ T cell ratio (Supplementary Table 1). The following fluorochrome-conjugated anti-human antibodies were used from BD Biosciences (San Jose, CA): CD3-FITC, CD3-PerCP-Cy5.5, CD4-APC, CD4-APC-Cy7, CD8-BV510, CD45RO-FITC, CD45RA-APC, CD45RA-APC-Cy7, CCR4-PE, CD27-PE, CD28-BV421, CD138-BV421, CCR6-BV421, CXCR3-PE, CCR7-A700, IL-17-BV786, IFN-γ-PE-Cy7, iso IgG1k-FITC, iso IgG1k-PE-Cy7, iso IgG2bk-APC, iso IgG1k-APC-Cy7, and iso IgG1k-BV510; from Biolegend (San Diego, CA): CD127-FITC, CD27-APC, CD57-PerCp-Cy5.5, CD19-BV510, PD-1-APC-Cy7, CXCR5-FITC, and TNFα-FITC; from eBioscience (San Diego, CA): CD4-PE-Cy7, and IL-2-PE; from Miltenyi Biotec (San Diego, CA): CD25-APC and KLRG1-PE; from Beckman Coulter (Brea, CA): CD38-PE-Cy7.

Hartzell S., Bin S., Cantarelli C., Haverly M., Manrique J., Angeletti A., Manna G.L., Murphy B., Zhang W., Levitsky J., Gallon L., Yu S.M, & Cravedi P. (2020). Kidney Failure Associates With T Cell Exhaustion and Imbalanced Follicular Helper T Cells. Frontiers in Immunology, 11, 583702.

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Profiling Immune Cell Phenotypes and Metabolic Parameters in Aging Adults

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Blood was collected from adults (n=6, four females and 2 males) aged 64-67 years in 2015. Peripheral blood mononuclear cells were isolated using a Ficoll–Hypaque density gradient (Amersham Biosciences) and centrifugation. Viable cells were counted, adjusted to 2×106/100 μL in 80% fetal bovine serum and 20% dimethylsulfoxide (Sigma), and frozen stored until the phenotyping. In 2021, cells were thawed, checked for viability, and stained with monoclonal antibodies to the T cell phenotypes CD4 PerCP Cy5.5, CD8 APC Cy7, CD27 APC, CD45RA PE; B cell phenotypes CD19 PE, CD27 APC, IgD PE Cy5.5 (eBioscience), and ACE CD143 fluorescein isothiocyanate (R&D Systems). After 30 minutes of incubation with monoclonal antibodies in the dark at 4 °C, the cells were washed with phosphate-buffered saline and centrifuged. Living cells (based on forward and side scatter) were acquired in the BD FACSCanto II flow cytometry system using the DIVA software (Becton Dickinson).
For assessing metabolic parameters, the serum of studied individuals was previously isolated through centrifugation and frozen stored until use. Measurement of metabolic parameters was performed in the Laboratório Central–Hospital São Paulo, Federal University of São Paulo.

Bueno V., Destro P.H., Teixeira D, & Frasca D. (2023). Angiotensin Converting Enzyme 1 Expression in the Leukocytes of Adults Aged 64 to 67 Years. JMIRx Med, 4, e45220.

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