Anti cd3 alexa fluor 700

Manufactured by Exbio

The Anti-CD3 Alexa Fluor 700 is a fluorescently labeled antibody that binds to the CD3 receptor on T cells. It can be used for the identification and analysis of T cell populations in flow cytometry applications.

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Lab products found in correlation

Permeabilization buffer, by Thermo Fisher Scientific (5 mentions) Fixation permeabilization buffer, by Thermo Fisher Scientific (5 mentions) Brefeldin a, by BioLegend (5 mentions) Anti cd4 ecd, by Beckman Coulter (4 mentions) Anti cd8 hv500, by BD (4 mentions) Anti ifn γ pe cy7, by Thermo Fisher Scientific (4 mentions) Anti granzyme b brilliant violet 421, by BD (3 mentions) Anti cd45 pe, by Exbio (2 mentions) Anti cd107a fitc monoclonal antibody, by BioLegend (2 mentions) Lsr fortessa analyzer, by Tree Star (1 mentions) Anti tim 3, by BioLegend (1 mentions) Anti tigit, by BioLegend (1 mentions) Anti pd 1, by Bristol-Myers Squibb (1 mentions) Anti cd56 alexafluor700, by BioLegend (1 mentions) Apc efluor 780, by Thermo Fisher Scientific (1 mentions) Anti perforin apc, by BioLegend (1 mentions) Anti cd45 percp, by Exbio (1 mentions) Anti cd56 percp cy5, by BioLegend (1 mentions) Phorbol myristate acetate (pma), by Merck Group (1 mentions)

5 protocols using anti cd3 alexa fluor 700

Functional T Cell Analysis in Tumor Samples

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Mononuclear cells isolated from fresh tumor specimens were stimulated with 50 ng/ml of phorbol 12-myristate 13-acetate (PMA) + 1 μg/ml of ionomycin for 1 h followed by 3 h incubation with brefeldin A (BioLegend). Unstimulated cells were used as a control. The cells were then washed in PBS, stained with anti-CD3 Alexa Fluor 700 (EXBIO), anti-CD4 ECD (Beckman Coulter) and anti-CD8 HV500 (BD Biosciences), fixed using fixation/permeabilization buffer (eBioscience), permeabilized with permeabilization buffer (eBioscience) and intracellularly stained with an anti-IFN-γ PE-Cy7 (eBioscience), anti-granzyme B Brilliant Violet 421 (BD Biosciences) (Additional file 1: Table S4). The percentage of CD3⁺CD8⁺ T cells producing IFN-γ and degranulating upon PMA/ionomycin stimulation were determined by flow cytometry. The data were analyzed with the FlowJo software package (Tree Star, Inc.). Gating strategy is depicted in Additional file 1: Figure S4.

Kasikova L., Hensler M., Truxova I., Skapa P., Laco J., Belicova L., Praznovec I., Vosahlikova S., Halaska M.J., Brtnicky T., Rob L., Presl J., Kostun J., Cremer I., Ryska A., Kroemer G., Galluzzi L., Spisek R, & Fucikova J. (2019). Calreticulin exposure correlates with robust adaptive antitumor immunity and favorable prognosis in ovarian carcinoma patients. Journal for Immunotherapy of Cancer, 7, 312.

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Characterizing Tumor-Infiltrating T Cells

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Mononuclear cells isolated from fresh tumor specimens (study group 2) were incubated with 10 µg/mL anti-PD-1 and 10 µg/mL anti-TIM-3 antibodies for 24 hours at 37°C and 5% CO₂. Thereafter, mononuclear cells were stimulated with 50 ng/mL phorbol 12-myristate 13-acetate (PMA)+1 µg/mL ionomycin for 1 hour followed by 3-hour incubation with brefeldin A (BioLegend). Unstimulated cells were used as control. Cells were washed in PBS, stained with anti-CD3 Alexa Fluor 700 (EXBIO), anti-CD4 ECD (Beckman Coulter), and anti-CD8 HV500 (BD Biosciences) monoclonal antibodies, fixed in fixation/permeabilization buffer (eBioscience), further permeabilized with permeabilization buffer (eBioscience) and intracellularly stained with an anti-IFN-γ PE-Cy7 (eBioscience) and anti-GZMB BV421 (BD Bioscience) monoclonal antibody. Flow cytometry was performed on the LSRFortessa analyzer, and data were analyzed with the FlowJo software package (Tree Star) (online supplementary figure 2).

Hensler M., Kasikova L., Fiser K., Rakova J., Skapa P., Laco J., Lanickova T., Pecen L., Truxova I., Vosahlikova S., Moserova I., Praznovec I., Drochytek V., Rehackova M., Brtnicky T., Rob L., Benes V., Pistolic J., Sojka L., Ryska A., Sautes-Fridman C., Fridman W.H., Galluzzi L., Spisek R, & Fucikova J. (2020). M2-like macrophages dictate clinically relevant immunosuppression in metastatic ovarian cancer. Journal for Immunotherapy of Cancer, 8(2), e000979.

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Assessing Immune Checkpoint Inhibitors in AML

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PBMCs from AML patients were incubated with 10 mg/mL anti-PD-1 (nivolumab, Bristol-Myers-Squibb), anti-TIM-3 (BioLegend) or anti-TIGIT (BioLegend) antibodies for 24 hours in 37°C and 5% CO₂, followed by incubation with 50 ng/mL PMA and 1 mg/mL ionomycin for 1 hour plus 3-hour incubation with brefeldin A (BioLegend). Unstimulated cells were used as control. Cells were then washed in PBS, stained with anti-CD45-PE (EXBIO), anti-CD3-AlexaFluor700 (EXBIO) and anti-CD56-PerCP-Cy5.5 monoclonal antibodies (BioLegend), fixed in fixation/permeabilization buffer (eBioscience), further permeabilized with permeabilization buffer (eBioscience) and intracellularly stained with anti-IFN-γ-PE-Cy7 antibodies (eBioscience).

Rakova J., Truxova I., Holicek P., Salek C., Hensler M., Kasikova L., Pasulka J., Holubova M., Kovar M., Lysak D., Kline J.P., Racil Z., Galluzzi L., Spisek R, & Fucikova J. (2021). TIM-3 levels correlate with enhanced NK cell cytotoxicity and improved clinical outcome in AML patients. Oncoimmunology, 10(1), 1889822.

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Assessing Immune Cell Activation in Tumor Samples

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Mononuclear cells isolated from fresh tumor specimens were stimulated with 50 ng/mL phorbol 12-myristate 13-acetate (PMA) + 1 μg/ml ionomycin in the presence of anti-CD107a FITC monoclonal antibody (BioLegend) for 1 h followed by 3 h incubation with brefeldin A (BioLegend). Unstimulated cells were used as control. Cells were then washed in PBS, stained with anti-CD45 PerCP (EXBIO), anti-CD3 Alexa Fluor 700 (EXBIO) or APC-eFluor780 (eBioscience), anti-CD4 ECD (Beckman Coulter), anti-CD8 HV500 (BD Biosciences) and anti-CD56 Alexa Fluor 700 (BioLegend) monoclonal antibodies, fixed in fixation/permeabilization buffer (eBioscience), permeabilized with permeabilization buffer (eBioscience) and stained with anti-IFN-γ PE-Cy7 (eBioscience), anti-granzyme B Brilliant Violet 421 (BD Biosciences) and anti-perforin APC (BioLegend) monoclonal antibodies. The percentage of CD3⁺CD8⁺ T cells and CD3⁻CD56⁺ NK cells producing IFN-γ and degranulating upon PMA/ionomycin stimulation were determined by flow cytometry. The data were analyzed with the FlowJo software package (Tree Star, Inc.).

Truxova I., Kasikova L., Hensler M., Skapa P., Laco J., Pecen L., Belicova L., Praznovec I., Halaska M.J., Brtnicky T., Salkova E., Rob L., Kodet R., Goc J., Sautes-Fridman C., Fridman W.H., Ryska A., Galluzzi L., Spisek R, & Fucikova J. (2018). Mature dendritic cells correlate with favorable immune infiltrate and improved prognosis in ovarian carcinoma patients. Journal for Immunotherapy of Cancer, 6, 139.

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Assessing NK Cell Functions in AML

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To assess NK cell functions in AML patients, fresh PBMCs were stimulated with 50 ng/mL phorbol 12-myristate 13-acetate (PMA, from Sigma Aldrich) plus 1 μg/mL ionomycin, or with K562 cells at 10:1 PBMC:tumor cell ratio plus overnight incubation with 200 U/mL IL-2 in the presence of anti-CD107a-FITC monoclonal antibody (BioLegend) for 1 h, followed by 3 h incubation with brefeldin A (BioLegend). Cells were then washed in PBS, stained with anti-CD45-PE (EXBIO), anti-CD3-AlexaFluor700 (EXBIO), anti-CD4-ECD (Beckman Coulter), anti-CD8-HV500 (BD Biosciences) and anti-CD56-PerCP-Cy5.5 (BioLegend) antibodies, fixed in fixation/permeabilization buffer (eBioscience), further permeabilized with permeabilization buffer (eBioscience) and intracellularly stained with anti-IFN-γ-PE-Cy7 (eBioscience) and anti-granzyme B-BrilliantViolet421 (BD Biosciences) antibodies.

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