Anti hla dr l243

Manufactured by BioLegend

Sourced in United States

Anti-HLA-DR (L243) is a monoclonal antibody that binds to the HLA-DR antigen. HLA-DR is a major histocompatibility complex class II (MHC-II) cell surface receptor that plays a crucial role in the immune system's antigen presentation process.

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6 protocols using anti hla dr l243

Immunophenotyping of Dendritic Cells

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Flow cytometry was performed as described and using gating strategies in ref. 32 . Briefly, for analysis of surface markers on iPS-DCs or Monocyte-derived DCs, cells were stained with Zombie Aqua fixable dye (BioLegend), Fc receptors were blocked using human TruStain FcX (BioLegend), and cells were then subsequently stained for iPS-DC surface markers anti-CD11c-FITC (Bu15, Biolegend) and anti-CD141-APC (M80, Biolegend) or Monocyte-derived DCs surface markers anti-HLA-DR (L243, Biolegend) and anti-CD209 (DC-SIGN) (9E9A8, Biolegend). For the detection of TLR2, cells were also stained anti-TLR2-PE (TL2.1, Biolegend) or TLR2-APC (W1514C, Biolegend) prior to fixation with 4% paraformaldehyde. Data were acquired using an Attune NxT flow cytometer (Thermo Fisher). Electronic compensation was performed with antibody (Ab) capture beads (BD Biosciences) stained separately with individual monoclonal antibodies used in the experimental panel. Data were analysed using the FlowJo software (TreeStar, Inc.).

Clement M., Forbester J.L., Marsden M., Sabberwal P., Sommerville M.S., Wellington D., Dimonte S., Clare S., Harcourt K., Yin Z., Nobre L., Antrobus R., Jin B., Chen M., Makvandi-Nejad S., Lindborg J.A., Strittmatter S.M., Weekes M.P., Stanton R.J., Dong T, & Humphreys I.R. (2022). IFITM3 restricts virus-induced inflammatory cytokine production by limiting Nogo-B mediated TLR responses. Nature Communications, 13, 5294.

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Single-Cell Sorting of Tumor-Infiltrating T Cells

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T cells were isolated from tumor single cell suspensions by antibody staining followed by cell sorting on a 5-laser FACSAria Fusion sorter (Stanford FACS Facility) purchased using funds from the Parker Institute for Cancer Immunotherapy. Tumor cell suspensions were stained in PBS with Zombie Aqua dye (Biolegend) for viability assessment. This was followed by staining in PBS with 2% FBS in Fc Blocking solution (Biolegend) plus the following antibodies: anti-CD4 (OKT4, Biolegend), anti-CD8 (SK1, Biolegend), anti-CD3 (OKT3, Biolegend), anti-CD45 (H130, Biolegend), anti-CD25 (BC96, Biolegend), anti-PD-1(EH12.2H7, Biolegend), anti-CD137 (4B4-1, BD Biosciences), anti-HLA-DR (L243, Biolegend). CD3⁺CD45⁺AquaZombie^- cells were index sorted directly into 96-well plates preloaded with 4 uL of capture buffer, snap frozen on dry ice, and stored at −80°C. Ectopic HLA-B^∗35 was detected with anti-HLA-BC monoclonal antibody (clone B1.23.2, Thermo Fisher Scientific). Transduced Jurkat 76 cells expressing exogenous TCRα/β chains were sorted on a FACSAria Fusion sorter at Stanford or a BD Biosciences Influx High Speed Cell Sorter at the Flow Cytometry Core Facility of the Cancer Institute of New Jersey.

Chiou S.H., Tseng D., Reuben A., Mallajosyula V., Molina I.S., Conley S., Wilhelmy J., McSween A.M., Yang X., Nishimiya D., Sinha R., Nabet B.Y., Wang C., Shrager J.B., Berry M.F., Backhus L., Lui N.S., Wakelee H.A., Neal J.W., Padda S.K., Berry G.J., Delaidelli A., Sorensen P.H., Sotillo E., Tran P., Benson J.A., Richards R., Labanieh L., Klysz D.D., Louis D.M., Feldman S.A., Diehn M., Weissman I.L., Zhang J., Wistuba I.I., Futreal P.A., Heymach J.V., Garcia K.C., Mackall C.L, & Davis M.M. (2021). Global analysis of shared T cell specificities in human non-small cell lung cancer enables HLA inference and antigen discovery. Immunity, 54(3), 586-602.e8.

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Comprehensive Immune Cell Profiling

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For surface staining, cells were washed once with FACS buffer (PBS containing 0.1% BSA and 0.02% sodium azide) and cells were incubated for 15 min with the antibody mixture in FACS buffer. Cell surface staining was performed using the following antibodies: anti-CD3 (UCHT1, eBioscience™), anti-CD4 (RPA-T4, BioLegend, San Diego, USA), anti-CD8 (RPA-T8, BioLegend), anti-CD56 (HCD56, BioLegend), anti-HLA-DR (L243, BioLegend), anti-CD107a (H4A3, BioLegend), and anti-CD253/Tumor Necrosis Factor Related Apoptosis Inducing Ligand (TRAIL) (RIK-2, BD). The Fixable Viability Dye (FVD) eFluor™ 780 (eBioscience™) was used to exclude dead cells from the analysis. Cells were washed with FACS buffer and fixated with 4% PFA for up to 2 h. Cells were washed twice with Intracellular Staining Perm Wash Buffer (BioLegend) and incubated for 20 min with anti-GzmB (GB11, BD) and anti-IFNγ (XNG1.2, BD) in Intracellular Staining Perm Wash Buffer. Cells were washed again twice with Intracellular Staining Perm Wash Buffer, collected in FACS staining buffer, and stored at 4°C until acquisition. Samples were acquired with a BD LSR II flow cytometer with a HTS module and data were analyzed using FACSDiva and FlowJo Version 10.8 (both BD Becton Dickinson, Heidelberg, Germany).

Karakoese Z., Schwerdtfeger M., Karsten C.B., Esser S., Dittmer U, & Sutter K. (2022). Distinct Type I Interferon Subtypes Differentially Stimulate T Cell Responses in HIV-1-Infected Individuals. Frontiers in Immunology, 13, 936918.

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Decidua Cell Immunophenotyping by Flow Cytometry

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For multi-parameter flow cytometry (LSR Fortessa, BD Biosciences, San Diego, CA), a cocktail of conjugated antibodies validated for the rhesus macaque was used (http://www.nhpreagents.org/NHP/clonelist.aspx?ID=15). For the majority of immunophenotyping studies, we used freshly isolated purified decidua cell suspensions. The following mAbs were used, all at a dilution of 1:20: anti-CD3 (SP34-2, anti-CD56 (NCAM16.2), anti-CD45 (D058-1283), (BD Biosciences); anti-HLA-DR (L243) (Biolegend, San Diego, CA); anti-CD14 (TUK14) (Life Technologies); anti-CD88 (P12/1) (AbD Serotec, Hercules, CA). Doublets were excluded on the basis of forward scatter properties and dead cells were excluded using LIVE/DEAD Fixable Aqua Dead Cell Stain (Life Technologies). Unstained and isotype control were used to determine positive staining for each marker. Data were analyzed using FlowJo software 9.5.2 (TreeStar Inc., Ashland, OR).

Coffey L.L., Keesler R.I., Pesavento P.A., Woolard K., Singapuri A., Watanabe J., Cruzen C., Christe K.L., Usachenko J., Yee J., Heng V.A., Bliss-Moreau E., Reader J.R., von Morgenland W., Gibbons A.M., Jackson K., Ardeshir A., Heimsath H., Permar S., Senthamaraikannan P., Presicce P., Kallapur S.G., Linnen J.M., Gao K., Orr R., MacGill T., McClure M., McFarland R., Morrison J.H, & Van Rompay K.K. (2018). Intraamniotic Zika virus inoculation of pregnant rhesus macaques produces fetal neurologic disease. Nature Communications, 9, 2414.

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PBMC Stimulation and Cytokine Analysis

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PBMCs were cultured in complete RPMI in flat-bottom 96 well plates, at 37°C in a 5% CO₂ atmosphere. 500,000 PBMCs were stimulated for 18 h with lysate of infected (iRBCs) or uninfected RBCs (uRBCs) in a ratio of 3 RBCs per PBMC, in the presence of anti-human leukocyte antigen HLA-DQ (SPVL-3; Beckmann Coulter, USA), anti-HLA-DP (B7/21; abcam, USA) and anti-HLA-DR (L243; Biolegend, USA), or in the presence of respective isotype controls. Following stimulation, cells were centrifuged and supernatants were recovered for cytokine analysis.

Portugal S., Moebius J., Skinner J., Doumbo S., Doumtabe D., Kone Y., Dia S., Kanakabandi K., Sturdevant D.E., Virtaneva K., Porcella S.F., Li S., Doumbo O.K., Kayentao K., Ongoiba A., Traore B, & Crompton P.D. (2014). Exposure-Dependent Control of Malaria-Induced Inflammation in Children. PLoS Pathogens, 10(4), e1004079.

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Multiparametric Flow Cytometry of Immune Cells

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Single cells from sputum were blocked with anti-CD16/CD32 (BD Biosciences, Franklin Lake, NJ) and stained with fluorochrome-labeled mAbs directed against cell-surface markers for 1 hour at 48C. For analysis of human induced sputum, the following antibodies were used. Anti-CD45 (HI30) was purchased from BD Biosciences. Anti-CD3e (UCHT1), anti-CD11c (3.9), anti-CD11b (ICRF44), anti-CD14 (HCD14), anti-CD15 (W6D3), anti-CD19 (HIB19), anti-CD49b (P1E6-C5), anti-CD68 (Y1/82A), anti-CD117 (104D2), anti-CD127 (A019D5), anti-CD206 (15-2), anti-FcεRIa (AER-37), anti-HLA-DR (L243), and anti-NKp44 (P44-8) were from BioLegend (San Diego, Calif). Anti-ST2L (B4E6) was from MD Bioproducts (Oakdale, Minn). For fluorescence-activated cell sorting of ILCs and alveolar macrophages (AMs) from mouse lungs, the following antibodies were used. Anti-CD45 (30-F11) and anti-ST2 (RMST2-33) were purchased from eBioscience (San Diego, Calif). Anti-CD11c (HL3), anti-CD11b (M1/70), anti-CD3e (145-2C11), anti-CD19 (ID3), and anti-CD49b (DX5) were from BD Biosciences. Anti-F4/80 (BM8), anti-FcεRIa (MAR-1), anti-CD127 (A7R34), and anti-CD25 (PC61) were from BioLegend. Flow cytometry was performed with the BD LSR II, BD LSRFortessa, and BD LSRFortessa X-20 and analyzed by using FlowJo (version 10.2) software (TreeStar, Ashland, Ore).

Kim J., Chang Y., Bae B., Sohn K.H., Cho S.H., Chung D.H., Kang H.R, & Kim H.Y. (2019). Innate immune crosstalk in asthmatic airways: Innate lymphoid cells coordinate polarization of lung macrophages. The Journal of allergy and clinical immunology, 143(5).

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