Hla dr clone l243

Manufactured by BioLegend

Sourced in United States

HLA-DR (clone L243) is a monoclonal antibody that recognizes the human leukocyte antigen (HLA) class II DR molecule. It is a useful tool for the identification and characterization of HLA-DR-expressing cells.

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Lab products found in correlation

Cd3 clone sp34 2, by BD (2 mentions) Cd8 clone rpa t8, by BioLegend (2 mentions) Cd206 clone 15 2, by BioLegend (2 mentions) Ab183322, by Abcam (2 mentions) Confocal microscopy system, by Zeiss (2 mentions) Nb600 633, by Novus Biologicals (2 mentions) Ccr2 7a7, by Abcam (2 mentions) Cd34 q bend1, by Abcam (2 mentions) Cd64 ab119843, by Abcam (2 mentions) Biotinconjugated anti mouse or anti rabbit secondary antibodies, by Vector Laboratories (2 mentions) Abc elite, by Vector Laboratories (2 mentions) Tunel staining, by Roche (2 mentions) Cd11c clone 3, by BioLegend (2 mentions) Cd14 clone m5e2, by BD (2 mentions) Cd4 clone rpa t4, by BioLegend (2 mentions) Triton x 100, by Merck Group (2 mentions) Cd80 clone 2d10, by BioLegend (2 mentions) Pe conjugated anti cd3 clone sp34, by BD (1 mentions) Cd4 clone okt4, by BioLegend (1 mentions) Lsrfortessa flow cytometer, by BD (1 mentions)

12 protocols using hla dr clone l243

Flow Cytometry Analysis of T Cells and Monocytes

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All CD11b⁺ myeloid cells were labeled with PE-conjugated anti-CD3 clone SP34 (BD Bioscience) and fluorescein isothiocyanate-conjugated anti-CD11b clone Bear1 (Beckman Coulter, Brea, CA) to assess the selection efficiency. Purified CD4⁺ T cells were stained with antibodies for HLA-DR clone L243 (BioLegend), CD3 clone SP34-2 (BD Bioscience), CD4 clone OKT4 (BioLegend), CD8 clone RPA-T8 (BioLegend), and TCRγδ clone B1.1 (eBioscience). Cells were stained for 20 min at room temperature in 100 μl phosphate-buffered saline–2% FBS and fixed for 10 min with Fix/Lyse buffer (Becton Dickinson, Franklin Lakes, NJ). After fixation, samples were analyzed in a BD LSRFortessa flow cytometer using DIVA software (Becton Dickinson, Franklin Lakes, NJ). The gating of CD3⁺ T cells was easily visualized as small CD3⁺ nonautofluorescent cells. All data were analyzed using FlowJo software. CD4⁺ T cell and monocyte counts were analyzed as previously described (38 (link)).

Avalos C.R., Price S.L., Forsyth E.R., Pin J.N., Shirk E.N., Bullock B.T., Queen S.E., Li M., Gellerup D., O'Connor S.L., Zink M.C., Mankowski J.L., Gama L, & Clements J.E. (2016). Quantitation of Productively Infected Monocytes and Macrophages of Simian Immunodeficiency Virus-Infected Macaques. Journal of Virology, 90(12), 5643-5656.

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Quantifying Endothelial Cell Surface Proteins

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Surface protein expression was quantified using flow cytometry. Briefly, endothelial cells were harvested, washed in 1% BSA in PBS, and incubated with an antibody against HLA-DR (clone L243, Biolegend) for 30 min on ice. After antibody incubation, endothelial cells were washed three times using 2% BSA in PBS and analyzed on a Attune NxT Flow Cytometer. Other endothelial cell antibodies used for flow cytometry include HLA-A,B,C (Biolegend) and CD31 (Biolegend). All results were analyzed using the FlowJo software (FlowJo LLC).

Cui J., Qin L., Zhang J., Abrahimi P., Li H., Li G., Tietjen G.T., Tellides G., Pober J.S, & Mark Saltzman W. (2017). Ex vivo pretreatment of human vessels with siRNA nanoparticles provides protein silencing in endothelial cells. Nature Communications, 8, 191.

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Characterization of Macrophage Surface Markers

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For analysis, MDM were detached from the PLLA films by incubation with TrypLE™ Express solution (Life Technologies) at 37°C for 10 min. For the analysis of surface markers, MDM were incubated for 20 minutes with saturating concentrations of monoclonal antibodies against HLA-DR (clone L243, Biolegend), CD206 (clone 15-2, Biolegend) and CD163 (clone GHI/61, Biolegend). To assess phagocytic activity, fluorescent yellow-green latex beads (1.0 µm mean particle size, Sigma-Aldrich) were incubated with Mϕ at a 1:10 (cell/beads) ratio for 45 min or 4 hours at 37°C. Before acquisition, cells were washed with FACS buffer (PBS with 2% FBS) to remove non-phagocytized beads. Samples were acquired on a LSRII flow cytometer with FACS Diva software (BD Biosciences). All data were analyzed using FlowJo v10 software (TreeStar Inc., Ashland, USA). Figure SI2 depicts the gating strategy for the characterization of macrophage surface markers.

Correia C.R., Gaifem J., Oliveira M.B., Silvestre R, & Mano J.F. (2017). Influence of surface modified poly(L-lactic acid) films on the differentiation of human monocytes into macrophages. Biomaterials science, 5(3), 551-560.

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Multiparametric Analysis of Tissue Sections

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Paraffin embedded sections were dewaxed in xylene, rehydrated,
endogenous peroxide activity quenched in 10% methanol and 3%
hydrogen peroxide, processed fro antigen retrieval by boiling in citrate
buffer pH 6.0 containing 0.1% Tween-20, blocked in 1% BSA,
and stained with the following primary antibodies overnight at 4 degrees C:
CD68 (KP1 eBiosceince 1:2000), CCR2 (7A7 Abcam 1:2000), CD34 (Q/bend1 Abcam
1:2000), Collagen 1 (COL-1 Abcam 1:2000), IL-1β (NB600-633 NOVUS
1:2000), Ki67 (ab15580 Abcam 1:1000), CD14 (ab183322 Abcam 1:2000), CD64
(ab119843 Abcam 1:4000), iNOS (ab76198 Abcam 1:1000), HLA-DR (clone L243
Biolegend 1:1000). The primary antibody was detected using a biotin
conjugated anti-mouse or anti-rabbit secondary antibodies (Vector Labs) in
conjunction with streptavidin HRP (ABC Elite, Vector Labs). The PerkinElmer
Opal Multicolor IHC system was utilized to visualize antibody staining per
manufacturer protocol. TUNEL staining (Roche) was performed per
manufacturer’s protocol. Immunofluorescence was visualized on a
Zeiss confocal microscopy system. Macrophages were quantified by examining
at least 4 similarly oriented sections from 4 independent samples in blinded
fashion.

Bajpai G., Schneider C., Wong N., Bredemeyer A., Hulsmans M., Nahrendorf M., Epelman S., Kreisel D., Liu Y., Itoh A., Shankar T.S., Selzman C.H., Drakos S.G, & Lavine K.J. (2018). The Human Heart Contains Distinct Macrophage Subsets with Divergent Origins and Functions. Nature medicine, 24(8), 1234-1245.

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Monocyte-Derived Macrophage Polarization

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Human monocytes were isolated from leukocyte concentrate (buffy-coat) from healthy donors. Human PBMCs were obtained by the Ficoll-Paque density gradient centrifugation, and the CD14⁺ population was sorted using CD14 MicroBeads (Miltenyi) following the manufacturer's indications. Cells were plated at 2 × 10⁵ cells/cm² and cultured in an MLR medium supplemented with 20 ng/ml of macrophage colony-stimulating factor (M-CSF) (Miltenyi). After 7 days of culture, macrophages were activated with 100 ng/ml of lipopolysaccharides (LPS) and cultured alone, with naïve hAD-MSC or pretreated hAD-MSC at a ratio of 1 : 10 for 24 h. To assess macrophage polarization, cells were harvested using Versene (Gibco) and incubated with 100 μM of Fc block (BD Bioscience) following the manufacturer's indications. Then, cells were stained using a cell viability marker (Life Technologies) and antibodies against CD86 (clone 2331 Fun-1), CD163 (clone GHI/61) (BD Bioscience), CD68 (clone Y1/82A), CD206 (clone 15-2), and HLA-DR (clone L243) (BioLegend, San Diego, California, USA). Data were acquired with a BD FACSCanto and analyzed using the FlowJo software (v.10) (TreeStar Inc.).

Tejedor G., Boisguerin P., Vivès É., Jorgensen C., Guicheux J., Vinatier C., Gondeau C, & Djouad F. (2022). PPARβ/δ-Interfering Peptide Enhanced Mesenchymal Stromal Cell Immunoregulatory Properties. Stem Cells International, 2022, 5494749.

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Phenotyping and Transcriptional Profiling of Immune Cells in Rheumatoid Arthritis

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Ex vivo and cultured PBMC were stained with APC-H7 (Tonbo Biosciences) or Brilliant Violet 405 (Molecular Probes) viability dye in the presence of different panels of monoclonal antibodies against lineage (Lin) (CD3 clone HIT3a, CD19 clone SJ25C1, CD20 clone 2H7, and CD56 clone 5.1H11), CD14 clone M5E2, CD16 clone 3G8, CD40 clone 5C3, CD86 clone IT2.2, ILT4 clone 42D1, HLA-DR clone L243, CD11c clone 3.9, CD1c clone L161, CD141 clone M80, CD64 clone 10.1, and PDL1 clone 29E.2A3 (BioLegend). Samples were analyzed on a Fortessa cytometer (BD Biosciences) at Centro Nacional de Investigaciones Cardiovasculares (CNIC, Madrid, Spain). Analysis of individual and multiparametric flow cytometry data was performed using FlowJo software (Tree Star Inc.).
For the transcriptional studies, viable human Lin^–CD14^–CD11c⁺HLADR⁺CD1c⁺ cDC, CD14^–CD11c⁺HLADR⁺CD141⁺ cDC, and total CD14⁺ Mo were sorted using a FACS Aria II sorter (BD Biosciences) from either PBMC from n = 4 untreated patients with RA and n = 4 HC, or SF from n = 3 individuals with RA and n = 3 suffering mechanic CPPD crystal–associated arthritis.

Delgado-Arévalo C., Calvet-Mirabent M., Triguero-Martínez A., Vázquez de Luis E., Benguría-Filippini A., Largo R., Calzada-Fraile D., Popova O., Sánchez-Cerrillo I., Tsukalov I., Moreno-Vellisca R., de la Fuente H., Herrero-Beaumont G., Ramiro A., Sánchez-Madrid F., Castañeda S., Dopazo A., González Álvaro I, & Martin-Gayo E. (2022). NLRC4-mediated activation of CD1c+ DC contributes to perpetuation of synovitis in rheumatoid arthritis. JCI Insight, 7(22), e152886.

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Detecting DPP4 Expression on Human Immune Cells

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In order to detect which subset of human immune cells expresses DPP4, freshly isolated human PBMCs were incubated with a cocktail of fluorescence labelled monoclonal antibodies, i.e. anti-CD3 (clone SP34-2, BD Biosciences); CD56 (clone MEM188, Serotec); CD20 (clone B9E9, Beckman-Coulter); HLA-DR (clone L243, Biolegend); CD14 (clone M5E2, BD Biosciences); DPP4 (clone 222113, R&D Systems); and live/dead cell viability marker (Thermo Fisher Scientific). Appropriate Ig isotype control antibodies were used as negative controls for each staining. S1 protein of MERS-CoV that recognizes DPP4, used in a previous study^{25 (link)}, was applied to compare DPP4 expression in camel and human PBMC. Cells were analyzed on a Canto II flow cytometer (BD Biosciences) and using FlowJo® software (FlowJo), then subsequently presented as mean percentage of positive cells.

Haverkamp A.K., Lehmbecker A., Spitzbarth I., Widagdo W., Haagmans B.L., Segalés J., Vergara-Alert J., Bensaid A., van den Brand J.M., Osterhaus A.D, & Baumgärtner W. (2018). Experimental infection of dromedaries with Middle East respiratory syndrome-Coronavirus is accompanied by massive ciliary loss and depletion of the cell surface receptor dipeptidyl peptidase 4. Scientific Reports, 8, 9778.

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Multiparametric Analysis of Tissue Sections

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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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Comprehensive Immune Profiling by Flow Cytometry

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Antibodies for CD4 (clone RPA-T4, Biolegend), CD8 (clone RPA-T8, Biolegend), CD11c (clone Bly6, BD Biosciences), CD40 (clone 5C3, Biolegend), CD80 (clone 2D10, Biolegend), CD86 (clone BU63, Biolegend), HLA-DR (clone L243, Biolegend), Galectin 9 (clone 9M1-3, Biolegend), PDL1 (clone), CD25 (clone BC96, Biolegend), FOXP3 (clone 206D, Biolegend), ILT3 (clone ZM4.1, Biolegend), ILT4 (clone 42D1, Biolegend), PD1 (clone EH12, BD Biosciences), Eomes (clone WD1928, eBioscience), IDO (clone eyedio, eBioscience) were obtained, and cells were stained according to manufacturer-recommended protocols. Events were collected on an LSRFortessa cytometer (BD), and data were analyzed in FlowJo (Treestar). Representative gating strategies are presented in Supplemental Figure 1.

Brusko M.A., Stewart J.M., Posgai A.L., Wasserfall C.H., Atkinson M.A., Brusko T.M, & Keselowsky B.G. (2020). Immunomodulatory Dual-Sized Microparticle System Conditions Human Antigen Presenting Cells Into a Tolerogenic Phenotype In Vitro and Inhibits Type 1 Diabetes-Specific Autoreactive T Cell Responses. Frontiers in Immunology, 11, 574447.

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