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Cd19 clone hib19

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CD19 (clone HIB19) is a mouse monoclonal antibody that recognizes the human CD19 antigen. CD19 is a cell surface molecule expressed on B lymphocytes and plays a role in B cell activation and differentiation.

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Lab products found in correlation

Cd4 clone rpa t4, by BioLegend (3 mentions) Software, by FlowJo (3 mentions) Cd127 clone a019d5, by BioLegend (2 mentions) Cd14 clone m5e2, by BioLegend (2 mentions) Cd66b clone g10f5, by BD (2 mentions) V500 anti cd45 clone hi30, by BD (2 mentions) Anti cd3 fitc clone ucht1, by BioLegend (2 mentions) Apc h7 anti cd16 clone 3g8, by BD (2 mentions) Af700 anti hla dr clone l243, by BD (2 mentions) Anti cd11c apc clone b ly6, by BD (2 mentions) Pe cy7 anti cd123 clone 7g3, by BD (2 mentions) Cd20 clone 2h7, by BioLegend (2 mentions) Lsrfortessa, by BD (2 mentions) Cd3 clone okt3, by BioLegend (2 mentions) Cd8 clone sk1, by BioLegend (2 mentions) Cd25 clone bc96, by BioLegend (2 mentions) Cd45ro clone uchl1, by BioLegend (2 mentions) Facsaria 2, by BD (2 mentions) Rnalater, by Thermo Fisher Scientific (2 mentions) Cd11c clone bu15, by BioLegend (1 mentions)

12 protocols using cd19 clone hib19

1

Isolation and Phenotyping of Human ILCs

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Lung cells from HSPC-engrafted MISTRG mice were isolated as described above. Cell surface staining with fluorescent antibodies was performed as previously described [17 (link), 18 (link)]. The following antibodies were used for flow cytometry: CD127 (clone A019D5, Biolegend), CRTH2 (clone BM16, Biolegend), CD94 (clone DX22, Biolegend), CXCR4 (clone 12G5, Biolegend), CXCR6 (clone K041E5, Biolegend), GITR (clone 108-17, Biolegend), CD16 (clone 3G8, Biolegend), CD69 (clone FN50, Biolegend), CD117 (104D2D1, Beckman Coulter), HLA-DR (clone G46-6, BD Biosciences), CD2 (clone RPA-2.10, BD Biosciences), CD56 (clone NCAM16.2, BD Biosciences), CD81 (clone JS-81, BD Biosciences), CD94 (clone HP-3D9, BD Biosciences), CD98 (clone UM7F8, BD Biosciences), CD103 (clone Ber-ACT8, BD Biosciences), and CD45RA (clone HI100, BD Biosciences). In all experiments, ILCs were defined as human CD45+Lin-CD3−TCRαβ−CD127+CD94− cells. Lineage markers included CD14 (clone M5E2, Biolegend), CD19 (clone HIB19, Biolegend), CD11c (clone Bu15, Biolegend), CD123 (clone 6H6, Biolegend), FcεRIα (clone AER-37 (CRA-1), Biolegend), CD34 (clone 581, BD Biosciences), TCRαβ (clone IP26) and CD3 (clone SK7).
Alisjahbana A., Mohammad I., Gao Y., Evren E, & Willinger T. (2023). Single-cell RNA sequencing of human lung innate lymphoid cells in the vascular and tissue niche reveals molecular features of tissue adaptation. Discovery Immunology, 2(1), kyad007.
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2

Quantifying Human Leukocyte Reconstitution

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Human leukocyte reconstitution was assessed by flow cytometry using Trucount (BD Biosciences, San Jose, CA, USA) enumeration to calculate the absolute number of human B cells, CD4+ and CD8+ T cells, monocytes, NK cells, and neutrophils per μL of blood [35 (link)]. Anti-human CD45 (clone H130, BD Biosciences, San Jose, CA, USA) and anti-mouse CD45 (clone 30-F11, BD Biosciences, San Jose, CA, USA) antibodies were used to differentiate mouse from human leukocytes. Human CD45+ cells were phenotyped using antibodies specific for human CD3 (clone UCHT1, Beckman Coulter, Brea, CA, USA), CD4 (clone RPA-T4, Biolegend, San Diego, CA, USA), CD14 (clone TüK4, Invitrogen, Carlsbad, CA, USA), and CD19 (clone HIB19, Biolegend, San Diego, CA, USA). Data analysis was performed using FlowJo software (v.9.9.4, FlowJo LLC, Ashland, OR, USA).
Maidji E., Moreno M.E., Rivera J.M., Joshi P., Galkina S.A., Kosikova G., Somsouk M, & Stoddart C.A. (2019). Cellular HIV Reservoirs and Viral Rebound from the Lymphoid Compartments of 4′-Ethynyl-2-Fluoro-2′-Deoxyadenosine (EFdA)-Suppressed Humanized Mice. Viruses, 11(3), 256.
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3

Phenotypic Characterization of Colonic and Immune Cells

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Single cell suspensions from surgical excess colon tissue or HIV-uninfected control PBMCs were stained for viability with a LIVE/DEAD fixable violet dead cell stain kit (Life Technologies). For phenotypic characterization of epithelial and tissue cells, cell surface antigens were stained as described above with the following mouse anti-human monoclonal antibodies: APC anti-CD326 (clone EBA-1; BD Bioscience), v500 anti-CD45 (clone HI30; BD Bioscience). For phenotypic characterization of monocytes and DCs from PBMCs, cell surface antigens were stained for 15 min at room temperature with the following mouse anti-human monoclonal antibodies: FITC anti-CD3 (clone UCHT1; Biolegend), CD19 (clone HIB19; Biolegend), CD66b (clone G10F5; BD Bioscience), v500 anti-CD45 (clone HI30; BD Bioscience), APC-H7 anti-CD16 (clone 3G8; BD Bioscience), AF700 anti-HLA-DR (clone L243; BD Bioscience), APC anti-CD11c (clone B-ly6; BD Bioscience), PE-Cy7 anti-CD123 (clone 7G3; BD Bioscience), PerCP-Cy5.5 anti-CD14 (clone M5E2; Biolegend). Stained cells were resuspended in PBS with 5 mM EDTA and sorted using a FACS Aria II (BD Biosciences).
Corleis B., Lisanti A.C., Körner C., Schiff A.E., Rosenberg E.S., Allen T.M., Altfeld M, & Kwon D.S. (2017). Early type I Interferon response induces upregulation of human β-defensin 1 during acute HIV-1 infection. PLoS ONE, 12(3), e0173161.
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4

Phenotypic Profiling of Cryopreserved PBMCs

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Cryopreserved PBMCs from acute (n = 8) and HIV-uninfected control subjects (n = 9) were thawed and 0.5x106 cells were stained for viability with a LIVE/DEAD fixable blue or green dead cell stain kit (Life Technologies). For phenotypic characterization of monocytes and DCs, cell surface antigens were stained for 15 min at room temperature with the following mouse anti-human monoclonal antibodies: FITC anti-CD3 (clone UCHT1; Biolegend), CD19 (clone HIB19; Biolegend), CD56 (clone HCD56; Biolegend), CD66b (clone G10F5; BD Bioscience), BV605 anti-CD4 (clone SK3; BD Bioscience), v500 anti-CD45 (clone HI30; BD Bioscience), APC-H7 anti-CD16 (clone 3G8; BD Bioscience), PE-Cy5 anti-CXCR4 (clone 12G5; BD Bioscience), AF700 anti-HLA-DR (clone L243; BD Bioscience), APC anti-CD11c (clone B-ly6; BD Bioscience), PE-Cy7 anti-CD123 (clone 7G3; BD Bioscience), BV421 anti-CCR5 (clone 2D7; BD Bioscience), PerCP-Cy5.5 anti-CD14 (clone M5E2; Biolegend). The cells were fixed with 2% paraformaldehyde before running on a LSR Fortessa flow cytometer (BD Biosciences) within 4 h. Flow data were analyzed with FlowJo (TreeStar).
Corleis B., Lisanti A.C., Körner C., Schiff A.E., Rosenberg E.S., Allen T.M., Altfeld M, & Kwon D.S. (2017). Early type I Interferon response induces upregulation of human β-defensin 1 during acute HIV-1 infection. PLoS ONE, 12(3), e0173161.
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5

PBMC Isolation and Flow Cytometry Analysis

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PBMCs were isolated from peripheral blood samples collected on days 0 (n = 9), 7 (n = 17), and 28 (n = 17) using Lymphoprep (Abbott Diagnostics, Lake Forest, IL, USA), and were stained with CD2 (clone RPA‐2.10; BioLegend, San Diego, CA, USA), CD3 (clones HIT3a and UCHT1; BioLegend), CD4 (clone RPA‐T4; BioLegend), CD10 (clone eBioCB‐CALLA; Thermo Fisher Scientific), CD19 (clone HIB19; BioLegend), CD20 (clone 2H7; BioLegend), CD27 (clone O323; Thermo Fisher Scientific), CD38 (clone HIT2; Thermo Fisher Scientific), and IgD (clone IA6‐2; BD Biosciences, San Jose, CA, USA) in the presence of human Fc receptor (FcR) block (Miltenyi Biotec, Bergisch Gladbach, Germany). CD19+ CD38++ CD27++ cells that were negative for CD2, CD3, CD4, CD10, IgD, and CD20 were counted as plasma cells.14 Data were acquired on an FACS Canto II (BD Biosciences) and analyzed using FlowJo software (FlowJo, Ashland, OR, USA).
Ainai A., van Riet E., Ito R., Ikeda K., Senchi K., Suzuki T., Tamura S., Asanuma H., Odagiri T., Tashiro M., Kurata T., Multihartina P., Setiawaty V., Pangesti K.N, & Hasegawa H. (2020). Human immune responses elicited by an intranasal inactivated H5 influenza vaccine. Microbiology and Immunology, 64(4), 313-325.
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6

Immune Cell Profiling of Peripheral Blood

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PB was obtained in EDTA-containing tubes. Peripheral blood mononuclear cells (PBMCs) were isolated using Lymphocyte Separation Medium (Corning) density gradient centrifugation. Fresh or cryopreserved PBMCs were incubated with anti-human IgG (Sigma). These antibodies were used for flow cytometry or cell sorting: anti-human CD3 (clone UCHT1; BioLegend), CD4 (clone RPA-T4; BD Biosciences), CD8 (clone RPA-T8; BioLegend), CD45 (clone HI30; BD Biosciences), CD56 (cone HCD56: BioLegend), CD19 (clone HIB19 BioLegend), and CX3CR1 (clone 2A9–1; BioLegend) antibodies. Samples were acquired using LSRFortessa (BD Biosciences), and data were analyzed with FlowJo software v10.1.5 (FlowJo LLC).
Abdelfatah E., Long M.D., Kajihara R., Oba T., Yamauchi T., Chen H., Sarkar J., Attwood K., Matsuzaki J., Segal B.H., Dy G.K, & Ito F. (2023). Predictive and Prognostic Implications of Circulating CX3CR1+ CD8+ T Cells in Non–Small Cell Lung Cancer Patients Treated with Chemo-Immunotherapy. Cancer Research Communications, 3(3), 510-520.
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7

Neoantigen-reactive CD4+ T-cell Isolation

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Cryopreserved PBMC were thawed and cultured at 2×106 cells/mL in a 24-well plate with anti-CD40 at 500 ng/mL and each of the five neoantigen peptides as well as a control HIV NEF (RYPLTFGWCF) peptide at 1 µg/mL overnight at 37°C. After 16 hours, cells were stained for with LIVE/DEAD Fixable Violet Dead Cell Stain (Invitrogen), followed by lineage markers CD14 (clone M5E2, Biolegend), CD19 (clone HIB19, Biolegend), CD4 (clone SK3, eBioscience), CD8 (clone 3B5, Invitrogen) and activation-induced markers CD69 (clone L78, BD Biosciences), CD137 (clone 4-1BB, Biolegend) and CD154 (clone TRAP1, BD Bioscience). Single neoantigen-reactive CD4 cells were sorted into RNAlater (Thermo Fisher) based on coexpression of CD69, CD137 and CD154 on a BD FACSAria II (BD Biosciences).
Church C., Pulliam T., Longino N., Park S.Y., Smythe K.S., Makarov V., Riaz N., Jing L., Amezquita R., Campbell J.S., Gottardo R., Pierce R.H., Choi J., Chan T.A., Koelle D.M, & Nghiem P. (2022). Transcriptional and functional analyses of neoantigen-specific CD4 T cells during a profound response to anti-PD-L1 in metastatic Merkel cell carcinoma. Journal for Immunotherapy of Cancer, 10(9), e005328.
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8

Multiparametric Immunophenotyping of PBMCs

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Whole blood samples were collected in cell preparation tubes with sodium citrate (BD Biosciences). PBMCs were obtained by centrifugation and viably frozen until analysis. PBMCs were thawed, washed with flow buffer (5% BSA, 2 mM EDTA in PBS) and incubated with LIVE/DEAD Fixable Aqua Dead Cell Stain (Life Technologies), Fc receptor blocking agent (Miltenyi Biotec) and stained with surface antibodies (CD3 clone OKT3, CD4 clone RPA-T4, CD8 clone SK1, CD14 clone HCD14, CD19 clone HIB19, CD25 clone BC96, ICOS clone C398.4A, HLA-DR clone L243, PD-1 clone 29F.1A12 all from BioLegend) for 20 min at 4°C. For Foxp3 and Ki67 staining, cells were fixed and permeabilized using a Fix/Perm buffer (eBiosciences) according to the manufacturer’s instructions, then stained with anti-Foxp3 (clone 206D, BioLegend) or anti-Ki67 antibody (clone B56, BD Biosciences).
For global protein acetylation analysis, after surface staining, cells were fixed with 0.4% paraformaldehyde (Thermo Fisher Scientific), permeabilized in Triton X-100 (Sigma-Aldrich) and subsequently stained with anti-acetylated lysine antibody (clone 15G10, BioLegend).
Hellmann M.D., Jänne P.A., Opyrchal M., Hafez N., Raez L.E., Gabrilovich D., Wang F., Trepel J.B., Lee M.J., Yuno A., Lee S., Brouwer S., Sankoh S., Wang L., Tamang D., Schmidt E., Meyers M.L., Ramalingam S.S., Shum E, & Ordentlich P. (2020). Entinostat plus pembrolizumab in patients with metastatic NSCLC previously treated with anti-PD-(L)1 therapy. Clinical cancer research : an official journal of the American Association for Cancer Research, 27(4), 1019-1028.
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9

Comprehensive T cell Phenotyping Protocol

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All samples were analyzed with an LSR Fortessa (BD Bioscience) and data were analyzed using FlowJo software (FlowJo LLC). T cell phenotype was evaluated via:Zombie Yellow Fixable Viability Kit (BioLegend), human CD45 (clone HI30, BioLegend), mouse CD45 (clone 30F11, BioLegend), CD3 (clone OKT3, BioLegend), CD4 (clone A161A1, BioLegend), CD8 (clone SK1, BioLegend), CD19 (clone HIB19, BioLegend), CD27 (clone M-T271, BioLegend), CD62L (DREG-56, BioLegend), PD-1 (clone MIH4, BD Bioscience), TIM-3 (clone F38-2E2, BioLegend), CD45RO (clone UCHL1, BioLegend), CD45RA (HI100, BioLegend).
Wang Y., Cho J.W., Kastrunes G., Buck A., Razimbaud C., Culhane A.C., Sun J., Braun D.A., Choueiri T.K., Wu C.J., Jones K., Nguyen Q.D., Zhu Z., Wei K., Zhu Q., Signoretti S., Freeman G.J., Hemberg M, & Marasco W.A. (2024). Immune-restoring CAR-T cells display antitumor activity and reverse immunosuppressive TME in a humanized ccRCC mouse model. iScience, 27(2), 108879.
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10

Comprehensive Immune Cell Profiling

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Cell staining of whole blood was performed for 25 min on ice in the dark in staining buffer composed of PBS, 0.5% BSA, and 0.05% sodium azide. Red blood cells were lysed by addition of 1× BD Pharm Lyse Buffer (BD Biosciences) and incubation in the dark at room temperature for 15 min. Following one wash, cells were fixed in 2% paraformaldehyde for 15 min at room temperature, washed again, and resuspended in staining buffer. A minimum of 300,000 total events were collected on a FACS Calibur using Cell Quest and analyzed with FlowJo software (TreeStar). Anti-human CD20 (clone 2H7, catalog #302304), CD19 (clone HIB19, catalog #302234), CD38 (clone HB7, catalog #356608), CD3 (clone UCHT1, catalog #300426), CD4 (clone RPA-T3, catalog #300506), CD25 (clone BC96, catalog #302632), CD127 (clone A019D5, catalog #351325), CCR4 (clone TG6, catalog #335405), CXCR5 (clone TG2, catalog #335001), CD45RO (clone UCHL1, catalog #304218), and PD-1 (clone EH12.2.H7, catalog #329907) antibodies were purchased from BioLegend, anti-human CD27 (clone O323, catalog #12-0279-42) from eBioscience, and anti-human ICOS antibody (clone DX29, catalog #557802) from BD Biosciences.
Panwar B., Schmiedel B.J., Liang S., White B., Rodriguez E., Kalunian K., McKnight A.J., Soloff R., Seumois G., Vijayanand P, & Ay F. (2021). Multi–cell type gene coexpression network analysis reveals coordinated interferon response and cross–cell type correlations in systemic lupus erythematosus. Genome Research, 31(4), 659-676.
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