Cd19 pecy7

Manufactured by Thermo Fisher Scientific

Sourced in United States

CD19-PECy7 is a fluorescently labeled antibody that binds to the CD19 surface marker. It is designed for use in flow cytometry applications to identify and enumerate CD19-positive cells.

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19 protocols using cd19 pecy7

Multiparametric Immune Cell Phenotyping

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Antibodies for surface staining included CCR7 APC-Cy7 (clone G043H7; Biolegend), CCR7 APC-eFluor780 (clone 3D12; eBioscience), CD4 PE-Cy5.5 (clone S3.5; Invitrogen), CD8 BV711 (clone RPA-T8; Biolegend), CD8 Qdot 605 (clone 3B5; Invitrogen), CD14 BV510 (clone M5E2; Biolegend), CD14 Pacific Blue (clone M5E2; custom), CD14 PE-Cy5 (clone 61D3; Abcam), CD14 PE-Cy7 (clone HCD14; Biolegend), CD16 Pacific Blue (clone 3G8; custom), CD16 PE-Cy5 (clone 3G8; Biolegend), CD16 PE-Cy7 (clone 3G8; Biolegend), CD19 BV510 (clone HIB19; Biolegend), CD19 Pacific Blue (clone HIB19; custom), CD19 PE-Cy5 (clone HIB19; Biolegend), CD19 PE-Cy7 (clone HIB19; Invitrogen), CD45RO ECD (clone UCHL1; Beckman Coulter), CD45RO PE-CF594 (clone UCHL1; BD Biosciences), CD107a PE-Cy5 (clone eBioH4A3; eBioscience), CD107a PE-Cy7 (clone H4A3; Biolegend), and HLA-DR Pacific Blue (clone LN3; Invitrogen). Antibodies for intracellular staining included CD3 BV570 (clone UCHT1; Biolegend), CD3 BV650 (clone OKT3; Biolegend), CD3 Qdot 585 (clone OKT3; custom), CD3 Qdot 650 (clone S4.1; Invitrogen), Eomes Alexa 647 (WD1928; eBioscience), Eomes eFluor 660 (WD1928; eBioscience), IFN-γ Alexa 700 (clone B27; Invitrogen), Perforin BV421 (clone B-D48, Biolegend), Perforin Pacific Blue (clone B-D48; custom), Perforin PE (clone B-D48, Cell Sciences), T-bet FITC (clone 4B10; Biolegend), and T-bet PE (clone 4B10; eBioscience).

Demers K.R., Makedonas G., Buggert M., Eller M.A., Ratcliffe S.J., Goonetilleke N., Li C.K., Eller L.A., Rono K., Maganga L., Nitayaphan S., Kibuuka H., Routy J.P., Slifka M.K., Haynes B.F., McMichael A.J., Bernard N.F., Robb M.L, & Betts M.R. (2016). Temporal Dynamics of CD8+ T Cell Effector Responses during Primary HIV Infection. PLoS Pathogens, 12(8), e1005805.

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Evaluating B Cell Cytokine Profiles in Multiple Sclerosis

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B cell cytokine production was evaluated in MS patients (RRMS, n = 45; PMS, n = 18) and HC (n = 20) incubating PBMCs for 24 h with CpG (10 μg/ml), adding in the last 4 h PMA (50 ng/mL) and ionomycin (1 μg/ml, Sigma-Aldrich), monensin (GolgiStop), and brefeldin (GolgiPlug, BD Biosciences). Samples were stained using CD19-PE-Cy7 (Invitrogen) and CD3-perCP (BD Biosciences), permeabilized, fixed, and stained with the following cytokine-specific monoclonal antibodies: anti-GM-CSF-APC, anti-IL-6-FITC (Biolegend), anti-IL-10-PE (BD Biosciences), and anti-TNFα conjugated with the CFP fluorochrome. Samples were acquired in LSR II Fortessa and data analyzed by FlowJo software, defining the proportions of B cells stained for specific cytokines.

Zabalza A., Vera A., Alari-Pahissa E., Munteis E., Moreira A., Yélamos J., Llop M., López-Botet M, & Martínez-Rodríguez J.E. (2020). Impact of cytomegalovirus infection on B cell differentiation and cytokine production in multiple sclerosis. Journal of Neuroinflammation, 17, 161.

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Phenotyping NCC-expressing immune cells

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Healthy control PBMCs were isolated and stained for NCC (rabbit anti-SLC12A3 polyclonal primary antibody; Life technologies, cat# PA5-80004; goat anti-rabbit APC conjugated secondary antibody; Invitrogen, cat#A-10931), CD4 (CD4-FITC), CD8 (PE-Cy7), and CD45RA (CD45RA-AF700), and analysed by FACS. Separate staining was performed for NCC (APC), CD45 (CD45-FITC; BioLegend, cat#304006), CD3 (CD3-BV711), and CD56 (CD56–BV510; BioLegend, cat#318340), in addition to NCC (APC) with CD19 (CD19-PE/Cy7; Invitrogen, cat#25-0199042). The percentage of each cell type expressing NCC was determined.

Evans R.D., Antonelou M., Sathiananthamoorthy S., Rega M., Henderson S., Ceron-Gutierrez L., Barcenas-Morales G., Müller C.A., Doffinger R., Walsh S.B, & Salama A.D. (2020). Inherited salt-losing tubulopathies are associated with immunodeficiency due to impaired IL-17 responses. Nature Communications, 11, 4368.

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Comprehensive Immune Cell Profiling

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Anti-mouse antibodies were purchased from BioLegend, eBioscience or Thermo Fisher for staining, including: CD3-BV510 (145-2C11), IL-7Rα-BV605 (A7R34), ICOS-PerCP.Cy5.5 (C398.4A), CD5-PE.Cy7 (53-7.3), CD8-PE.Cy7 (53-6.7), CD19-PE.Cy7 (eBio1D3), NK1.1-PE.Cy7 (PK136), CD11b-PE.Cy7 (M1/70), CD11c-PE.Cy7 (N418), Gr-1-PE.Cy7 (RB6-8C5), Fcϵ1-PE.Cy7 (MAR-1), TER-119-PE.Cy7 (TER-119), Siglec-F-Alexa Fluor 647 (E50-2440) and Live/Dead-A780. For flow cytometry, single-cell suspensions were generated and antibodies used at 1:200 for staining. Data were acquired using LSRFortessa flow cytometer and FACSDiva software, and analysed using FlowJo software.

Lok L.S., Walker J.A., Jolin H.E., Scanlon S.T., Ishii M., Fallon P.G., McKenzie A.N, & Clatworthy M.R. (2021). Group 2 Innate Lymphoid Cells Exhibit Tissue-Specific Dynamic Behaviour During Type 2 Immune Responses. Frontiers in Immunology, 12, 711907.

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Comprehensive Immune Cell Profiling

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Flow cytometric analyses were performed as described (48 (link)). The following Abs were used: TLR4/MD-2 Complex APC (Thermo Fisher Scientific, 17–9924-82), F4/80 Antigen PE (Thermo Fisher Scientific, 12–4801-80), F4/80 Antigen PE Cy7 (Thermo Fisher Scientific, 25–4801-82), CD11b PE Cy7 (Biolegend, 101215), CD11b eFluor 450 (Thermo Fisher Scientific, 48–0112-82), CD11b APC (Thermo Fisher Scientific, 17–0112-82), CD45 PerCP Cy5.5 (Thermo Fisher Scientific, 45–0451-82), CD45 APC (Thermo Fisher Scientific, 17–0451-82), Ly6G APC Cy7 (Tonbo Biosciences, 25–1276), CD19 PE Cy7 (Thermo Fisher Scientific, 25–0193-82), CD4 eFluor 450 (Thermo Fisher Scientific, 48–0042-82), TCRβ (Thermo Fisher Scientific, 12–5961-83), CD8 Alexa Fluor 488 (Thermo Fisher Scientific, 53–0081-82), CD11c PerCP Cy5.5(Thermo Fisher Scientific, 45–0114-82), CD115 APC (Thermo Fisher Scientific, 17–1152-82), Fc Block (Thermo Fisher Scientific, 14–9161-71) and a Zombie Aqua Fixable Viability kit (Biolegend, 423101).

Rosen D.A., Seki S.M., Fernández-Castañeda A., Beiter R.M., Eccles J.D., Woodfolk J.A, & Gaultier A. (2019). Modulation of the Sigma-1 Receptor-IRE1 pathway is beneficial in preclinical models of inflammation and sepsis.. Science translational medicine, 11(478), eaau5266.

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Multiparameter Flow Cytometry Immunophenotyping

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Cells were labelled with allophycocyanin (APC) CD11c (HL3), BV421 CD11c (HL3),
FITC CD80 (16-10A1), CD86 PE (GL1), APC CD40 (1C10), PerCP-eFluor 710 CD40
(1C10), PE-Cy7 CD19 (1D3), PerCP-Cy5.5 (H1.2F3), APC TCRβ (H57-597),
phycoerythrin (PE) TCRβ (H57-597), FITC CD3 (145-2c11), APC-eFluor 780
major histocompatibility complex II (M5/114.15.2), BV605 CD45.1 (A20), BV786
CD45.2 (104) and APC IFNγ (XMG1.2) purchased from eBioscience or BD
Pharmingen. pS6 analysis used phospho-S6 Ser 235/236 (Cell Signaling
Technologies) and secondary was PE-conjugated donkey anti-rabbit immunoglobulin
G (Jackson ImmunoResearch). 2-NBDG (Life Technologies) was added to cells at
35 μM for 1 h prior to analysis. Live cells were
gated by forward scatter (FSC-A) and side scatter (SSC-A) analysis. Single cells
were selected by FSC-A and FSC-W analysis. For intracellular staining, cells
were then fixed and permeabilized using Cytofix/Cytoperm reagent (BD
Pharmingen). For cytokine analysis, endocytosis was blocked using golgi plug (BD
Pharmingen) for 4 h. Gating strategies for all flow cytometry
analysis are outlined in Supplementary
Figs 5 and 6. Data were acquired on a FACSCanto or LSRFortessa (Becton
Dickinson) and analysed using the FlowJo software (TreeStar).

Lawless S.J., Kedia-Mehta N., Walls J.F., McGarrigle R., Convery O., Sinclair L.V., Navarro M.N., Murray J, & Finlay D.K. (2017). Glucose represses dendritic cell-induced T cell responses. Nature Communications, 8, 15620.

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Multiparameter Flow Cytometry Immunophenotyping

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The surface markers included FITC‐CD3 (MA1‐7640, eBioscience, San Diego, CA, USA), APC‐Cy7‐CD4 (100413, Biolegend, San Diego, CA, USA), APC‐CD4 (100411, Biolegend), PerCP‐CD8 (100,731, Biolegend), PE‐Cy7‐CD8 (100721, Biolegend), PE‐Foxp3 (12‐5773‐82, eBioscience), APC‐Helios (17‐9883‐42, eBioscience), PerCP‐CD44 (103035, Biolegend), FITC‐CD62L (104405, Biolegend), FITC‐lin (22‐7770‐72, eBioscience), PerCP‐CD34 (50‐0341‐82, eBioscience), APC‐Sca‐1 (17‐5981‐82, eBioscience), PE‐Sca‐1 (12‐5981‐82, eBioscience), APC‐c‐kit (17‐1171‐82, eBioscience), PE‐c‐kit (12‐1171‐82, eBioscience), PE‐B220 (12‐0452‐82, eBioscience), FITC‐IgM (11‐5790‐81, eBioscience), PE‐Cy7‐CD19 (12‐0193‐82, eBioscience), APC‐CD19 (17‐0193‐82, eBioscience), PE‐Cy7‐CD16/32 (25‐0161‐82, eBioscience), PE‐Cy7‐CD45 (25‐0451‐82, eBioscience), PerCP‐5.5‐CD45.2 (45‐0454‐82, eBioscience), and APC‐Cy7‐CD45.1 (17‐0453‐82, eBioscience) obtained from Invitrogen. Intracellular staining with PE‐Foxp3 (72‐5775‐40, eBioscience) was performed with Foxp3 staining kits (Invitrogen, Carlsbad, CA, USA).

Chen J., Liu J, & Huang H. (2023). Lkb1 loss in regulatory T cells leads to dysregulation of hematopoietic stem cell expansion and differentiation in bone marrow. FEBS Open Bio, 13(2), 270-278.

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Isolation and Purification of Bone Marrow Cells

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Mouse hind limbs were collected and bisected by cutting through the knee joint. Muscle and connective tissues of both the femur and tibia were removed by scraping the diaphysis of the bone. After cleaning, bones were rinsed in ice-cold 70% ethanol for 10-15 min and washed in cold PBS for 5 min for three times. The femur and tibia were cut just at the end of the marrow cavity and the marrow was sluiced out by RPMI 1640 (Invitrogen) containing 10% FBS and 2 mM EDTA followed by filtering through 100 μm filter. Cells were centrifuged at 2 000× g for 10 min and red blood cells were lysed with RBC lysis buffer (Roche). Washed and collected cells from previous step were purified by 40%/80% percoll, and stained sequentially with Violet Dye and antibody cocktail for flow cytometry analysis. The antibody cocktail for different lineage markers included CD3e-PECy7 (eBioscience, 25-0031), CD19-PECy7 (eBioscience, 25-0193), CD11b-PECy7 (eBioscience, 25-0112), Ly6G-PECy7 (eBioscience, 25-5931), Ter119-PECy7 (eBioscience, 25-5921). The cells were stained spontaneously with an antibody against Kit/CD117-APC (eBioscience, 17-1171, 1:200).

Liu Q., Yang R., Huang X., Zhang H., He L., Zhang L., Tian X., Nie Y., Hu S., Yan Y., Zhang L., Qiao Z., Wang Q.D., Lui K.O, & Zhou B. (2015). Genetic lineage tracing identifies in situ Kit-expressing cardiomyocytes. Cell Research, 26(1), 119-130.

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Multi-color FACS Analysis of Spleen Cells

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Analysis of spleen cells was carried out using a multi-color FACS analysis, following a standard procedure (39 (link), 40 (link)). Cells were stained with a combination of directly conjugated mAbs, washed, and analyzed using FACSCanto II (BD Biosciences, San Jose, CA, USA). The antibodies used were CD3-FITC, CD4-APC, and CD8-APC-Cy7 (Biolegend, San Diego, CA, USA), CD19-PE-Cy7 and CD11c-PE (eBioscience), and CD11b-PE-Cy7 (BD Biosciences). Non-viable cells staining positive with 7AAD dye (eBioscience) were excluded from the analysis. Data collected on 50,000 cells were analyzed using FACSDiva software (BD Biosciences).

George J.A., Bashir G., Qureshi M.M., Mohamed Y.A., Azzi J., al-Ramadi B.K, & Fernández-Cabezudo M.J. (2016). Cholinergic Stimulation Prevents the Development of Autoimmune Diabetes: Evidence for the Modulation of Th17 Effector Cells via an IFNγ-Dependent Mechanism. Frontiers in Immunology, 7, 419.

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Comprehensive Cervical Cell Phenotyping

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The phenotype of cells collected on the cervical brushes was determined using flow cytometry. Squamous epithelial cells and dead leukocytes absorbed 4′,6-diamino-2-phenylindole (DAPI). Cells that excluded DAPI were examined using directly labelled monoclonal antibodies against the following human proteins: CD45-FITC and CD16-PerCPCy5.5 from BD Biosciences (Franklin Lakes, NJ, USA), CD163-PE, CD11b-PE, CD20-PE, CD14-PerCPCy5.5, HLA-DR-PECy7, CD19-PECy7, CD27-PECy7, CCR7-PECy7, CD11b-APC (activation epitope CBRM1/5) and CD16-APC from eBioscience (San Diego, CA, USA); CD235a-PE, CD68-PE, CD66b-PE, CD103-PE, CD11c-PE, CX3CR1-PE, CCR2-PerCPCy5.5, CD3-PECy7, CCR1-APC, CD4-APC, CD33-APC and CD64-APC from Biolegend (San Diego, CA, USA). Flow cytometric data were collected on an LSRII (BD Biosciences). A minimum of 300,000 events were collected for each group of antibodies so that even a leukocyte population consisting of 0.01% of the total host-derived cells could be reliably detected. Data were analysed using FlowJo 9.6.4 (TreeStar, Ashland, OR, USA).

Hunter P.J., Sheikh S., David A.L., Peebles D.M, & Klein N. (2016). Cervical leukocytes and spontaneous preterm birth. Journal of Reproductive Immunology, 113, 42-49.

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