Anti ctla4 pe

Manufactured by BioLegend

Sourced in United States

Anti-CTLA4-PE is a fluorescent-labeled antibody that specifically binds to the CTLA-4 (Cytotoxic T-Lymphocyte Antigen 4) protein. CTLA-4 is an immune checkpoint receptor expressed on the surface of T cells and plays a role in regulating T cell activation and immune responses.

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7 protocols using anti ctla4 pe

CTLA-4 Expression in Activated T Cells

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Total isolated PBMC (2 × 10⁵ cells, 96-well-round bottom plate; Greiner) were cultured in the presence of anti-CD3 monoclonal antibody (mAb, LEAF purified anti-human CD3, Clone HIT3a, 1 µg/ml, Biolegend), human IL-2 (Proleukin, Novartis, 0.1 µM) and sertraline (1 µM). To label (capture) the cell-surface-exposed CTLA-4, we added anti-CTLA-4 PE or a matching PE isotype control (both Biolegend) to the culture medium and incubated the cells at 37°C for 24 h. Afterwards, the cells were stained for the expression of CD4, CD25, Foxp3 and CD45RA. To determine the total CTLA-4 amount per cell, parallel samples were first fixed and permeabilized before staining for the expression of CTLA-4.

Wiese T., Dennstädt F., Hollmann C., Stonawski S., Wurst C., Fink J., Gorte E., Mandasari P., Domschke K., Hommers L., Vanhove B., Schumacher F., Kleuser B., Seibel J., Rohr J., Buttmann M., Menke A., Schneider-Schaulies J, & Beyersdorf N. (2021). Inhibition of acid sphingomyelinase increases regulatory T cells in humans. Brain Communications, 3(2), fcab020.

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Evaluating In Vitro Binding Capacity of Immune Checkpoint Proteins

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Stable cell lines were generated to evaluate in vitro binding capacity of anti-PD-1, CD80-Fc, and 4-1BBL-Fc proteins. For this purpose, total RNA was extracted from anti-CD3/CD28 activated human PBMCs, and the first-strand cDNA was synthesized by Thermo cDNA synthesis kit (Thermo Scientific, USA). Human PD-1 (hPD-1), CTLA-4 (hCTLA-4), CD28 (hCD28), and 4-1BB (h4-1BB) coding genes were amplified by PCR using specific primers and separately inserted into pCHO1.0 expression vector. As described earlier, these constructs were transfected to CHO-K1 by electroporation, and then stable cell lines were produced by puromycin/MTX strategy. The expression of these proteins on the surface of transfected CHO-K1 cells was screened by flow cytometry using anti-PD-1-PE (BioLegend™, USA), anti-CD28-PE (BD Pharmingen™, USA), anti-CTLA4-PE (BioLegend), and anti-4-1BB-PE (BioLegend™, USA) antibodies.

Kaviani E., Hosseini A., Mahmoudi Maymand E., Ramzi M., Ghaderi A, & Ramezani A. (2022). Triggering of lymphocytes by CD28, 4-1BB, and PD-1 checkpoints to enhance the immune response capacities. PLOS ONE, 17(12), e0275777.

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Multiparametric FACS Analysis of T Cells

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For fluorescence-activated cell sorting (FACS) analysis, cells were labelled with saturating amounts of antibodies in FACS buffer (PBS containing 0.1% BSA and 0.02% NaN₃) to stain cell-surface antigens for 15 min on ice, followed by a fixation/permeabilization step (Fix/Perm buffer, eBioscience) for 30 min at room temperature and intracellular staining for 45 min at room temperature in permeabilization buffer (eBioscience). Stained cells were analysed on an LSR II flow cytometer (BD Biosciences).
For the staining of PBMC, the following Abs were used: Anti-CD3-PE-Cy7, anti-CD4-PerCP, anti-CD4-Pacific Blue, anti-CD4-FITC, anti-CD4-Alexa Fluor 700, anti-CD8-PE, anti-CD25-APC, mIgG₁-APC, anti-CD45RA-PE, anti-CD45RA-PErCP-Cy5.5, anti-CD127-PE, anti-Foxp3-APC anti-Foxp3-Pacific Blue, anti-CCR7-Alexa Fluor 488, anti-Ki-67-Alexa Fluor 700, anti-CTLA-4-PE, anti-CTLA-4-PE-Cy7 (all Biolegend), anti-CD25-PE, anti-CD8-FITC, PE-Cy5-streptavidin, anti-CD86-biotin (all BD Bioscience) and viability dye (Thermo Fischer).
For di-4-ANEPPDHQ (ANE) staining, PBMC were incubated with 4 mM of ANE (Invitrogen) together with anti-CD4 APC-Cy7, anti-CD45RA BV510 and anti-CD25 APC (all from Biolegend) in RPMI medium for 30 min at 37°C and were immediately analysed by FACS.

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Comprehensive Flow Cytometry Immunophenotyping

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For flow cytometry analysis, tumor and blood samples were stained with a cocktail of monoclonal mouse anti-human conjugated antibodies (mAbs): anti-CD45-PercP (clone HI30), anti-CD3-PercP (HIT3a), anti-CD3-APC (UCHT1), anti-CD19-PE (HIB19), anti-CD15-PE (HI98), anti-CD161-FITC (HP-3G10), anti-CD4-FITC (OKT4), anti-CD8-PE (HIT8a), anti-HLA-DR-APC (L243), anti-CD127-PE-Cy7 (A019D5), anti-CD1c-APC-Cy7 (L161), anti-CD163-PE (GHI/61), anti-CD206-APC-Cy7 (15–2), anti-PD-1-FITC (EH12.2H7), anti-PD-L1-APC (29E2A3), anti-CTLA4-PE (L3D10), anti-CD69-APC-Cy7 (FN50), anti-Tim3-APC (F38-2E2), anti-IL-8-APC (E8N1), anti-IFN-γ-PE (4S.B3), anti-IFN-γ-APC-Cy7 (4S.B3), anti-Granzyme B-FITC (QA16A02), anti-IL-1β-FITC (JK1B-1), anti-IL-2-PE-Cy7 (MQ1-17H12), anti-IL-6-APC (MQ2-13A5), anti-IL-17-FITC (BL168), anti-IL-23/IL-12-PE (C11.5), anti-TGF-β-APC (TW4-6H10), all from Biolegend; anti-IDO-PE (eyedio) and anti-IL-10-FITC (BT-10), both from eBioscience; anti-CD25-PE (MEM-181) and anti-CD11b-FITC (LT11) from ImmunoTools.
The antibodies used for immunofluorescence were: mouse monoclonal anti-human CD8 (32-M4) from Santa Cruz Biotechnology and rabbit polyclonal anti-human HLA-DRA from Sigma Aldrich.

Saraiva D.P., Jacinto A., Borralho P., Braga S, & Cabral M.G. (2018). HLA-DR in Cytotoxic T Lymphocytes Predicts Breast Cancer Patients' Response to Neoadjuvant Chemotherapy. Frontiers in Immunology, 9, 2605.

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Isolation and Characterization of Regulatory T Cells

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PBMCs were isolated from peripheral blood by Ficoll gradient centrifugation (DAKEWE, Shenzhen), CD4⁺CD25^high Treg cells and CD8⁺ T cells were sorted from PBMCs by flow cytometry through signalling of appropriate antibodies: anti‐CD4‐FICT (Biolegend), anti‐CD25‐APC (Biolegend) and anti‐CD8‐Percp‐cy5.5 (Biolegend), respectively. The antibodies were incubated at 4°C for 30 minutes in the dark. Flow cytometric analyses were performed using Fluorescence Activated Cell Sorter (FACS) Canto II (BD Biosciences, Heidelberg, Germany), and the results were analysed with BD FACSDiva Software version 6.1.2. For detection of surface molecules of CD4⁺CD25^high regulatory cells, PBMCs were incubated with the appropriate mAb: anti‐CD4‐FITC (eBioscience) and anti‐CD25‐PE (eBioscience). For intracellular staining including the staining of Foxp3, HO‐1, IL‐10, LAP and CTLA‐4, cells were fixed, permeabilized and stained according to the manufacturer's instruction. The following mAb were used: anti‐Foxp3‐APC (eBioscience), anti‐HO‐1‐PE (Abcam), anti‐IL‐10‐PE (eBioscience), anti‐LAP‐PerCP (eBioscience) and anti‐CTLA‐4‐PE (Biolegend).

Zhang Q., Cui T., Chang Y., Zhang W., Li S., He Y., Li B., Liu L., Wang G., Gao T., Li C, & Jian Z. (2018). HO‐1 regulates the function of Treg: Association with the immune intolerance in vitiligo. Journal of Cellular and Molecular Medicine, 22(9), 4335-4343.

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Multiparametric Flow Cytometry Analysis

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Mouse or human cells were stained with the following antibodies at a dilution ratio of 1:50: anti-CD45 PerCP-Cy5.5 (BD biosciences, 550994), anti-CD11b FITC (Biolegend, 101206), anti-CD86 APC (Biolegend, 105011), anti-CD206 PE (Biolegend, 141706), anti-CD8a FITC (Biolegend, 100706), anti-CD4 APC (Biolegend, 100516), anti-MHCI PE (Biolegend, 114607), anti-Foxp3 PE (BD biosciences, 563101), anti-IFN-γ PE (BD biosciences, 554412), anti-TNF-α PE/Cyanine7 (Biolegend, 506324), anti-Granzyme B PE/Cyanine7 (Biolegend, 396410), anti-PD1 PE/Cyanine7 (Biolegend, 109110), anti-Tim3 PE (Biolegend, 134003), anti-CTLA4 PE (Biolegend, 106305) and anti-LAG3 PE (Biolegend, 125207). For intracellular staining, the cells were fixed in Fixation and Permeabilization kit (eBioscience, 00-5521-00). After washed with Fixation and Permeabilization buffer, the cells were stained intracellularly for 30 min in the dark. All samples were acquired on BD LSRFortessa (BD Biosciences) or ACEA NovoCyte (ACEA Biosciences), and were analyzed using FlowJo (FlowJo LLC) or NovoExpress software (ACEA Biosciences).

Ye Z., Ai X., Yang K., Yang Z., Fei F., Liao X., Qiu Z., Gimple R.C., Yuan H., Huang H., Gong Y., Xiao C., Yue J., Huang L., Saulnier O., Wang W., Zhang P., Dai L., Wang X., Wang X., Ahn Y.H., You C., Xu J., Wan X., Taylor M.D., Zhao L., Rich J.N, & Zhou S. (2023). Targeting Microglial Metabolic Rewiring Synergizes with Immune Checkpoint Blockade Therapy for Glioblastoma. Cancer discovery, 13(4), 974-1001.

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Flow Cytometry Immunophenotyping Protocol

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Following stimulation for flow cytometry experiments, cells were fixed with 4% para-formaldehyde for 10 min before being washed 3 times with PBS and blocked/quenched with 5% bovine serum + 0.1 mM glycine overnight at 4°C. Cells were stained with 1 μg/ml of anti-BTLA AlexaFluor 647 (RRID AB_2650979; BioLegend Cat. No. 344519), anti-CTLA4 PE (RRID AB_10645522; BioLegend Cat. No. 349905), anti-CD86 Brilliant Violet 421 (RRID AB_10899582; BioLegend Cat. No. 305425), anti-CD69 APC (RRID AB_314844; BioLegend Cat. No. 310909), or anti-CD25 FITC (RRID AB_314273; BioLegend Cat. No. 302603) for 1 h at room temperature then washed 3 times with PBS before being analysed using a FACSCanto II™ flow cytometer (BD Biosciences). Data were analysed using FlowJo version 8.8.7. Staining was performed in three independent experiments with cells from different donors.

Felce S.L., Farnie G., Dustin M.L, & Felce J.H. (2021). RNA-Seq analysis of early transcriptional responses to activation in the leukaemic Jurkat E6.1 T cell line. Wellcome Open Research, 5, 42.

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