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Cd19 pe cy5

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CD19-PE-Cy5 is a fluorescently-labeled antibody that binds to the CD19 surface antigen. It is used for the identification and enumeration of B cells in flow cytometry applications.

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Lab products found in correlation

Cd10 pacific blue, by BioLegend (4 mentions) Cd34 pe, by BioLegend (3 mentions) Qbsf 60, by Quality Biological (3 mentions) Cd33 apc, by BD (3 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions) Fetal bovine serum (fbs), by Atlanta Biologicals (3 mentions) Methocult h4434 classic, by STEMCELL (2 mentions) Eclipse ts100 inverted microscope, by Nikon (2 mentions) Efluor 780, by Thermo Fisher Scientific (2 mentions) Ack lysing buffer, by Thermo Fisher Scientific (2 mentions) Cd14 bv510, by BioLegend (1 mentions) Cd16 pe cy7, by BioLegend (1 mentions) Cd8 bv711, by BioLegend (1 mentions) Cd19 bv510, by BioLegend (1 mentions) Ccr7 apc cy7, by BioLegend (1 mentions) Cd19 pacific blue, by BioLegend (1 mentions) Cd16 pe cy5, by BioLegend (1 mentions) Cd19 pecy7, by Thermo Fisher Scientific (1 mentions) Cd45ro pe cf594, by BD (1 mentions) Cd107a pe cy5, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

CD19-PE (55 citations) Anti-CD19-PE/Cy5 (2 citations) CD19-PE/Cy5 (clone HIB19, (2 citations) PE/Cy5-conjugated rat anti-mouse CD19 (2 citations)

10 protocols using cd19 pe cy5

1

Immunophenotyping of Whole Blood Samples

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Venous blood was drawn into an ethylenediaminetetraacetic acid (EDTA) S-monovette (Sarstedt, Nümbrecht, Germany) for immunophenotyping. Whole blood (100 µl per flow cytometry panel) was directly stained in FACS tubes with fluorescence-labeled antibodies (BD-Biosciences, Franklin Lakes, United States: CD8-PerCP-Cy5.5, CD5-PE, CD45RA-PE-CF594, CD19-PE-Cy5, CD4-PE-Cy7, CD45RO-APC, CD38-A700, CD3-APC-H7, CD138-BV421, CD10-BV510, CD27-BV605, CD14-PE-CF594, CD19-PE-Cy5, CD25-PE-Cy7, CD16-A700, CD3-APC-H7, CD11b-BV421, HLADR-BV510; Biolegend, San Diego, United States: CD11c-BV605; Miltenyi Biotec, Bergisch-Gladbach, Germany: BDCA1-APC, BDCA2-PE) for 15 min in the dark at room temperature (RT). After washing, red blood cell (RBC) lysis was performed with 2 ml ACK lysing buffer (Thermo Scientific, Waltham, United States) for 7 min in the dark at RT. 2 ml of FACS buffer (phosphate buffered saline, 3% fetal calf serum, 1% sodium azide) was added to wash the samples twice. Subsequently, cells were resuspended in 400 µl FACS-buffer. After the staining procedure, cells were measured by flow cytometry (LSR Fortessa cytometer, BD Biosciences, Franklin Lakes, United States) and analyzed with FACS-Diva Software (BD Biosciences, Franklin Lakes, United States). The gating strategy is available in the supplementary data section of this paper (Supplementary Figure S7).
Shalchi-Amirkhiz P., Bensch T., Proschmann U., Stock A.K., Ziemssen T, & Akgün K. (2023). Pilot study on the influence of acute alcohol exposure on biophysical parameters of leukocytes. Frontiers in Molecular Biosciences, 10, 1243155.
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2

CD34+ Hematopoietic Progenitor Differentiation

Cited 4 times
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Unstimulated and TLR-Stimulated CD34+ hematopoietic progenitors were plated at 10,000 cells/well for liquid, stromal cell-free B lineage cultures, or 1000 cells per sample in Methocult H4434 Classic (Stemcell Technologies), according to the manufacturer’s directions, as previously described [4 (link),5 (link),18 (link),19 (link)]. For B lineage liquid culture, cells were grown in QBSF®60 (Quality Biological, Inc.) supplemented with 10% FBS (Atlanta Biologicals), 100U Penicillin/Streptomycin (Gibco), 10% hASC conditioned media (LaCell LLC), 10 ng/ml stem cell factor, 10 ng/ml granulocyte stimulating factor, 5 ng/ml FLT-3 ligand, and 5 ng/ml IL-7 (as above). Half of the media was replaced once a week. After 4 weeks, cells were harvested, counted and assessed by flow cytometry using CD34-PE, CD10-Pacific Blue, CD19-PE-Cy5 (BioLegend), CD33-APC (BD Biosciences), and goat anti-ARID3a with a FITC-conjugated anti-goat secondary. Methocult cultures were incubated for 14 days and colonies were analyzed visually for monocytic, granulocytic and erythrocytic lineage cell colonies and counted using a Nikon Eclipse TS100 inverted microscope. Methocult cultures were harvested with warm 1X PBS, washed twice with warm PBS and once with ice cold PBS and then assessed by flow cytometry for the following surface markers: CD34, CD33, CD14, CD16, and CD41 with intracellular ARID3a.
Ratliff M.L., Shankar M., Guthridge J.M., James J.A, & Webb C.F. (2020). TLR engagement induces ARID3a in human blood hematopoietic progenitors and modulates IFNα production. Cellular immunology, 357, 104201.
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3

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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4

Multiparameter Flow Cytometry Analysis

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In all assays, cells were incubated with anti-FcγRII/IIIb before incubation with fluorochrome-conjugated antibodies. Dead cells were excluded by propidium iodide (PI) uptake or fixable viability dye eFluor780 (eBioscience), and gated on singlets. Whole blood was anticoagulated with EDTA and lysed with ACK lysis buffer before staining. Antibodies were obtained from eBioscience, BD, or BioLegend: CD3-FITC or Pacific Blue; CD4-APC, eFluor605, or eFluor650; CD19-PE-Cy5; MHC II-eFluor 650 or Alexa Fluor 700; F4/80-eFluor 450 or Pacific Blue; Siglec-F-PE; CD44-V500 or Alexa 700; B220-eFluor650 or V500; Ly6G-Alexa 700 or APC-Cy7; Ly6C-Alexa Fluor 700 or APC-Cy7; ST2-FITC or biotin; CD11b-PerCP-Cy5.5, Alexa Fluor 488, PE-Cy7, or Alexa Fluor 700; CD11c-APC, PerCP-Cy5.5, or PE-Cy7; CD69-PE-Cy7; CD45-eFluor605 or APC-eFluor780; CD103-APC, Brilliant Violet 421, or biotin; CD80-FITC; CD86-PE-Cy7, PerCP-Cy5.5, or eFluor605; OX40L-biotin; CCR7-biotin or APC; Streptavidin-APC, Qdot-800 (Invitrogen), or PE; IL-12p40-PE or APC; and TNF-PE or APC. Fluorescence minus one (FMO) controls were used for gating. Data were acquired on an LSR II (BD) and analyzed using FlowJo (Tree Star).
Chu D.K., Jimenez-Saiz R., Verschoor C.P., Walker T.D., Goncharova S., Llop-Guevara A., Shen P., Gordon M.E., Barra N.G., Bassett J.D., Kong J., Fattouh R., McCoy K.D., Bowdish D.M., Erjefält J.S., Pabst O., Humbles A.A., Kolbeck R., Waserman S, & Jordana M. (2014). Indigenous enteric eosinophils control DCs to initiate a primary Th2 immune response in vivo. The Journal of Experimental Medicine, 211(8), 1657-1672.
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5

Multiparameter Flow Cytometry of CSF Immune Cells

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Flow cytometry was conducted using an LSRFortessa (BD Biosciences). A panel consisting of antibodies conjugated to six different fluorophores was used to classify subsets of memory T cells and for drop-seq. Antibodies used were: CD8α-Pacific blue (BioLegend), CD3-BV650 (BD Biosciences), CD45RA-APC-Cy7 (BioLegend), CCR7–488 (Bio-Legend), IL-7Rα-PE (BioLegend) and CD27-PE-Cy7 (BioLegend). For characterization of CSF cells, this same panel was used, but CD19-PE-Cy5 (BioLegend) and CD14-Qdot-705 (Thermo Fisher Scientific) were included. For sorting CSF T cells for TCR plate-seq, the following antibodies were used: CD8α-PE (BioLegend), CD161-PE-Cy7 (BioLegend), CXCR3-APC (BioLegend), CD4-APC-Alexa700 (Thermo Fisher Scientific), CD39-APC-Cy7 (BioLegend), CD38-FITC (BioLegend), PD-1-BV421 (BD Biosciences), CD45RA-BV605 (BD Biosciences), CD3-BV650 (BD Biosciences), CD27-BV786 (BD Biosciences) and CD127-BUV395 (BD Biosciences). For each experiment, a compensation matrix was developed using singly stained and unstained controls or fluorescent beads, and all analysis was conducted in Cytobank.
Gate D., Saligrama N., Leventhal O., Yang A.C., Unger M.S., Middeldorp J., Chen K., Lehallier B., Channappa D., De Los Santos M.B., McBride A., Pluvinage J., Elahi F., Tam G.K., Kim Y., Greicius M., Wagner A.D., Aigner L., Galasko D.R., Davis M.M, & Wyss-Coray T. (2020). Clonally expanded CD8 T cells patrol the cerebrospinal fluid in Alzheimer’s disease. Nature, 577(7790), 399-404.
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6

Comprehensive Immune Profiling of Tumor Cells

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Tumor tissues were digested using Mouse Tumor Dissociation kits (Miltenyi) according to the manufacturer’s protocols. Isolated cells were stained for viability using the Aqua Amine fixable live dead dye (Thermo Fisher) using standard protocols. After viability staining, cells were resuspended in FACS buffer (PBS with 0.5% BSA and 2 mM EDTA) and Fc blocked using mouse or human TrueStain FcX (Biolegend). For analysis stains, cells were incubated for 30 minutes at 4C with the following anti-mouse antibodies from Thermo Fisher: CD90.2 (Thy-1.2) SuperBright 645, clone 53-2.1; CD4-AlexaFluor 488, clone GK1.5, CD8a-SuperBright 780, clone 53-6.7; CD3e-PE-Cy5, clone 145-2C11; B220-PE-Cy5, clone RA3-6B2; CD19-PE-Cy5, clone 1D3; CD11c-PE-eFluor610, clone N418; CD45-AlexaFluor 700, clone 30-F11; MHCII-APC-eFluor 780, clone M5/114.15.2; antibodies from Biolegend: NK1.1-PE-Cy5, clone PK136; F4/80-AlexaFluor488; XCR-1-PE, clone ZET; anti-mouse CD80-APC, clone 16-10A1; CD86-Bv785, clone GL-1, PD-1-PE, clone 29F.1A12; LAG-3-PE-Cy7, clone C987W; anti-human CD40-Bv421, clone 5C3; antibodies from BD: anti-mouse CD40-Bv421, clone 3/23; anti-mouse CD172a-Bv605 (Sirpα), clone P84. Samples were analyzed using an Attune NxT flow cytometer (Thermo Fisher) and data was analyzed using FlowJo 10 (TreeStar).
Garris C.S., Wong J.L., Ravetch J.V, & Knorr D.A. (2021). Intravesical dendritic cell targeting with Fc-enhanced CD40 agonistic antibodies induces durable bladder cancer immunity. Science translational medicine, 13(594), eabd1346.
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7

CD34+ Hematopoietic Progenitor Differentiation

Cited 4 times
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Unstimulated and TLR-Stimulated CD34+ hematopoietic progenitors were plated at 10,000 cells/well for liquid, stromal cell-free B lineage cultures, or 1000 cells per sample in Methocult H4434 Classic (Stemcell Technologies), according to the manufacturer’s directions, as previously described [4 (link),5 (link),18 (link),19 (link)]. For B lineage liquid culture, cells were grown in QBSF®60 (Quality Biological, Inc.) supplemented with 10% FBS (Atlanta Biologicals), 100U Penicillin/Streptomycin (Gibco), 10% hASC conditioned media (LaCell LLC), 10 ng/ml stem cell factor, 10 ng/ml granulocyte stimulating factor, 5 ng/ml FLT-3 ligand, and 5 ng/ml IL-7 (as above). Half of the media was replaced once a week. After 4 weeks, cells were harvested, counted and assessed by flow cytometry using CD34-PE, CD10-Pacific Blue, CD19-PE-Cy5 (BioLegend), CD33-APC (BD Biosciences), and goat anti-ARID3a with a FITC-conjugated anti-goat secondary. Methocult cultures were incubated for 14 days and colonies were analyzed visually for monocytic, granulocytic and erythrocytic lineage cell colonies and counted using a Nikon Eclipse TS100 inverted microscope. Methocult cultures were harvested with warm 1X PBS, washed twice with warm PBS and once with ice cold PBS and then assessed by flow cytometry for the following surface markers: CD34, CD33, CD14, CD16, and CD41 with intracellular ARID3a.
Ratliff M.L., Shankar M., Guthridge J.M., James J.A, & Webb C.F. (2020). TLR engagement induces ARID3a in human blood hematopoietic progenitors and modulates IFNα production. Cellular immunology, 357, 104201.
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8

Expansion of CD34+ Hematopoietic Progenitors

Cited 2 times
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CD34+ hematopoietic progenitors were isolated from mononuclear cells using the EasySep Human CD34 Positive Selection Kit (StemCell Technologies), and plated in triplicate at 10,000 cells per well as described (25 (link), 26 (link)). Briefly, cells were cultured in QBSF®60 (Quality Biological, Inc.) supplemented with 10% FBS (Atlanta Biologicals), 100U Penicillin/Streptomycin (Gibco), 10% hMSC conditioned media (graciously provided by Dr. J. Gimble) and containing stem cell factor, 10ng/ml granulocyte stimulating factor, 5ng/ml FLT-3 ligand, and 5ng/ml IL-7 (R&D). Half of the media was replaced once a week for the duration. After 4 weeks of culture, cells were harvested, counted and assessed for the following markers by flow cytometry: CD34 PE, CD10 Pacific Blue, CD19 PE-Cy5 (all from BioLegend), and CD33 APC (BD Biosciences). For proliferation assays, cells were labeled with CFSE (10μM, Invitrogen) in PBS for 10 minutes, followed by 4X volume of RPMI supplemented with 20% FBS for 5 minutes on ice to quench CFSE uptake. Cells were washed once with RPMI containing 20% FBS, and twice with PBS supplemented with 20% FBS prior to in vitro culture. After 6 days, cells were harvested and assessed for CD34 and CFSE expression levels via flow cytometry.
Ratliff M.L., Ward J.M., Merrill J.T., James J.A, & Webb C.F. (2014). Differential Expression of the Transcription Factor ARID3a in Lupus Patient Hematopoietic Progenitor Cells. Journal of immunology (Baltimore, Md. : 1950), 194(3), 940-949.
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9

B Cell Immunophenotyping by Flow Cytometry

Cited 1 time
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PBMCs were isolated from heparinized peripheral blood (~15 ml) with Ficoll-Paque Plus (GE Healthcare), and stained with the following fluorochrome-labeled antibodies: CD19 PE-Cy5, CD10 Pacific Blue (BioLegend), IgD PerCP-Cy5.5, CD27 PE-Cy7, CD38 Alexa Fluor 700 (BD Pharmingen), and IgM APC (Southern Biotech). PBMCs were fixed (3% paraformaldehyde) and permeabilized (0.1% Tween-20) prior to staining with goat anti-human ARID3a antibody [21 (link)] and a rabbit anti-goat IgG FITC secondary (Invitrogen). Gating for individual B cell subsets was described previously [3 (link)] and used with the following B (CD20+) cell subset markers: transitional (IgD+CD27−CD10+), naïve (IgD+CD27−CD10+), MZ-like Memory (IgD+CD27+), Memory (IgD+CD27+), and double-negative (DN) (IgD−CD27−) B cells. Non-B cells were excluded using the following markers on the fluorochrome, APC: T cells (CD3), Monocytes, macrophages, and granulocytes (CD14), NK cells, neutrophils, macrophage and dendritic cells (CD16), and NK and NKT cells (CD56). Isotype controls (Caltag, BD Pharmingen, and eBioscience) were used for gating. Data (500,000 events per sample) were collected using an LSRII (BD Biogenics) and FACSDiva (BD Biosciences) software version 4.1 and were analyzed using FlowJo (Tree Star) software version 9.5.2.
Ward J.M., Ratliff M.L., Dozmorov M.G., Wiley G., Guthridge J.M., Gaffney P.M., James J.A, & Webb C.F. (2016). Human effector B lymphocytes express ARID3a and secrete interferon alpha. Journal of autoimmunity, 75, 130-140.
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10

Phenotyping Immune Cell Markers

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Antibodies against the following proteins were used: β-Actin (Merck, Madrid, Spain; monoclonal clone AC-15; #A5441), ADAM10 (Abcam, Cambridge, UK; rabbit polyclonal; #ab1997), N-Cadherin (Merck; rabbit monoclonal; #04–1126), Calreticulin (Thermo-Fisher, Barcelona, Spain; mouse monoclonal; #MA5–11723), CD19-PE-Cy5 (Biolegend, San Diego, CA, USA; clone 6D5; # 115509), CD3ε- FITC (Biolegend; clone 145-2C11; # 100305), CD314 (NKG2D)-PerCP-eFluor 710 (Thermo-Fisher; clone CX5; #46–5882-80), CD335 (NKp46)-APC (Biolegend; clone 29A1.4; #137607), CD45-PerCP-Cy5.5 (Biolegend; clone 30–711; Cat# 103131), CD8a-PE (Thermo-Fisher; clone 53.6.7; # 12–0081-82), F4/80-APC (eBioscience, San Diego, CA, USA; clone BM8; #17–4801-30), FLAG (Merck; monoclonal clone M2; #F1804), HIF1α (Novus, St Charles, MO, USA; monoclonal clone H1alpha67; #NB100–105), MLANA (Abcam, Cambridge, UK; mouse monoclonal; #ab200544), MITF (Millipore; mouse monoclonal; #MAB3747), Rae1δ (Thermo-Fisher; mouse monoclonal; #14–5756-81), HLA-ABC (Thermo-Fisher; mouse monoclonal; #MA5–11723), HLA-E (Thermo-Fisher; mouse monoclonal; #14–9953-80).
Sánchez-del-Campo L., Martí-Díaz R., Montenegro M.F., González-Guerrero R., Hernández-Caselles T., Martínez-Barba E., Piñero-Madrona A., Cabezas-Herrera J., Goding C.R, & Rodríguez-López J.N. (2021). MITF induces escape from innate immunity in melanoma. Journal of Experimental & Clinical Cancer Research : CR, 40, 117.
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