Cd3 alexa fluor 700

Manufactured by BioLegend

Sourced in United Kingdom, Germany

CD3-Alexa Fluor 700 is a fluorescently-labeled antibody that binds to the CD3 antigen, which is expressed on the surface of T cells. The Alexa Fluor 700 dye is used to label the antibody, providing a fluorescent signal that can be detected and measured using flow cytometry or other fluorescence-based techniques.

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9 protocols using cd3 alexa fluor 700

T Cell Phenotyping and Sorting

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The antimouse TCRβ (mTCR)- PerCp-Cy5.5, CD8a-eFluor 450, were purchased from eBioscience. CD3-APC-Cy7, CD4- PE/FITC/APC, 41BB- APC/PE, CD8- PE-Cy7/FITC, OX40- FITC/PE-Cy7, CD62L-PE, and CD45RO-APC/BV421 were purchased from BD Biosciences. CD3-Alexa Fluor 700, CD62L-PE-Cy7, and the live/dead stains- DAPI/PI were purchased from BioLegend.
For FACS sample preparation, cells were harvested and washed with FACS buffer. Cells were resuspended in FACS buffer at a concentration of 1–50 × 10⁶ cells/mL and extracellular fluorescence-conjugated primary antibodies were added and mixed in. After a 20- to 60-minute incubation at 4°C, which was also protected from light exposure, the samples' cells were washed and resuspended with FACS buffer.
The flow cytometry assays were performed on FACSCanto I/II (BD Biosciences) and the acquired data were analyzed with FlowJo software (TreeStar). Cells were sorted to: Live/CD3⁺, separated for CD4⁺ or CD8⁺, T_EMRA/T_EM/T_CM based on sorting out CD62L⁺/CD45RO⁻ (naïve T cells). Cocultures were sorted for enrichment or into single reactive cells based on 41BB⁺/OX40⁺ (both double- and single-positive cells), Live/CD3⁺, separated for CD4⁺ or CD8⁺ by SH800S/MA900 instrument (Sony Biotechnology) or by FACSAria II (BD Biosciences).

Levin N., Paria B.C., Vale N.R., Yossef R., Lowery F.J., Parkhurst M.R., Yu Z., Florentin M., Cafri G., Gartner J.J., Shindorf M.L., Ngo L.T., Ray S., Kim S.P., Copeland A.R., Robbins P.F, & Rosenberg S.A. (2021). Identification and Validation of T-cell Receptors Targeting RAS Hotspot Mutations in Human Cancers for Use in Cell-based Immunotherapy. Clinical Cancer Research, 27(18), 5084-5095.

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Multiparametric Flow Cytometry Analysis

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PBMCs (1 × 10⁶) were Fc blocked (130-059-901; Miltenyi Biotec), stained, and acquired on a BD LSR II or a BD LSRFortessa (5 (link)). For intracellular cytokine staining, 1 × 10⁶ PBMCs were cultured in the presence of the appropriate antiegn (ColI or ColV) in DMEM containing 5% FBS at 37°C. After overnight incubation, the cells were stimulated with PMA (10 ng/ml) and ionomycin (1 μg/ml) for 5 h, with the addition of brefeldin A (420601; BioLegend) for the last 3 h. The following Abs were used: CD3-FITC or CD3–Alexa Fluor 700 (317306/100216; BioLegend or 557943; BD Biosciences), CD11b-BV421 (562632; BD Biosciences), CD56-allophycocyanin (555518; BD Biosciences), CD14–Alexa Fluor 700 (325614; BioLegend), CD16-allophycocyanin-H7 (560195; BD Biosciences), LAIR1-PE (550811; BD Biosciences), HLA-DR–PE-Cy7 (25-9956-41; eBioscience), CD123-BV421 (306017; BioLegend), lineage-BV510 (348807; BioLegend), CD11c-allophycocyanin (559877; BD Biosciences), PE-IgG₁ κ isotype control (55749; BD Biosciences), CD196/CCR6-FITC (11-1969-42; eBioscience), CD194/CCR4-PECy7 (561034; BD Biosciences), CD4-BUV395 (563552/563790; BD Biosciences), CD183/CXCR3-allophycocyanin (550967; BD Biosciences), IFN-γ-BV421 (563376; BD Biosciences), IL-17-PE (559502; BD Biosciences), γδ TCR-FITC (118105; BioLegend). Data were analyzed using FlowJo (Treestar).

Agashe V.V., Jankowska-Gan E., Keller M., Sullivan J.A., Haynes L.D., Kernien J.F., Torrealba J.R., Roenneburg D., Dart M., Colonna M., Wilkes D.S, & Burlingham W.J. (2018). Leukocyte-Associated Ig-like Receptor 1 Inhibits Th1 Responses but Is Required for Natural and Induced Monocyte-Dependent Th17 Responses. Journal of immunology (Baltimore, Md. : 1950), 201(2), 772-781.

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Comprehensive Immune Cell Phenotyping

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Cell surface and intracellular molecular expressions were evaluated by flow cytometry using CytoFLEX (BeckmanCoulter, U.S.A.). Fluorescein-conjugated mouse anti-human antibodies were used, including CD3-Alexa Fluor 700, CD3-BV650, CD8-BV786, CD8-PerCP/Cy5.5, CD45RA-APC/CY7, CCR7-PE/CY7,CD27-PE, CD28-BV421, CD69-APC/CY7, CD103-BV605, CXCR3-BV510, HLA-DR-APC, CD39-BV421, PD-1-PE, CD127-PE/CY7, CD62L-PE/CY7, Perforin-APC, Granzyme B-PE, IFN-γ-PE, and IL-4-APC (Biolegend, UK). For cell-surface staining, single-cell suspensions were stained on ice for 30 min in PBS with 1% fetal bovine serum (FBS). For intracellular staining, cells were fixed and permeabilized using the Fix/Perm kit (BD Biosciences, U.S.A.). To detect intracellular cytokines, CD8⁺T cells were stimulated for 6 h with phorbol 12-myristate 13-acetate (PMA; 1 μg/mL; Sigma) and ionomycin (1 μg/mL; Sigma), and 4 h with GolgiStop (1 μL/mL; BD Biosciences) in a round-bottom 96-well plate. Thereafter, cells were harvested, stained for surface expression, and then fixed and permeabilized for intracellular staining. Flow cytometry data was analyzed using FlowJo software (BD, UK) and CytoExpert software (Beckman Coulter, U.S.A.).

Liu L., Huang X., Xu C., Chen C., Zhao W., Li D., Li L., Wang L, & Du M. (2020). Decidual CD8+T cells exhibit both residency and tolerance signatures modulated by decidual stromal cells. Journal of Translational Medicine, 18, 221.

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Multiparametric Flow Cytometry of Cytokine-Producing T Cells

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Intracellular staining for the cytokines Interferon-γ (IFN-γ), Tumor necrosis factor α (TNF-α) and IL-2 was performed as originally mentioned by Jung et al. [59 (link)]. Briefly, cells after one week MLPC were restimulated with appropriate antigens in the presence of Brefeldin A for six hours (Biolegend, San Diego, CA, USA). Cells were harvested for live-dead NEAR (Invitrogen, Thermo Fisher Scientific, Waltham, MA USA) and surface marker staining followed by fixation using paraformaldehyde (PFA, Sigma Aldrich). For surface marker staining the following antibodies were used: CD3 Alexa fluor700 (Biolegend, Fell, Germany), CD8 Peridinin chlorophyll (PerCP, Biolegend), CD197 (CCR7) phycoerythrin Cy7 (eBioscience, San Diego, CA, USA) and CD45RA fluorescein isothiocyanate (FITC, BD Biosciences, Heidelberg, Germany). After permeabilization cells were blocked by FcR blocking reagent (Miltenyi Biotec) then stained for intracellular cytokines using anti-IFN-γ PE, anti-TNF-α Pacific Blue and anti-IL-2 AmCyan antibodies (BD Biosciences, Heidelberg, Germany). Samples were measured with the flow cytometer BD LSR II and analyzed using the BD FACSDiva analysis software (BD Biosciences).

Mani J., Wang L., Hückelhoven A.G., Schmitt A., Gedvilaite A., Jin N., Kleist C., Ho A.D, & Schmitt M. (2016). Definition and characterization of novel HLA-*A02-restricted CD8+ T cell epitopes derived from JCV polyomavirus with clinical relevance. Oncotarget, 8(2), 2485-2500.

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Comprehensive Immune Phenotyping Protocol

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The following anti-human antibodies were used for staining: CD3-FITC, CD3- Alexa Fluor 700, CD8a-Alexa Fluor700, CD4-Percp-Cy5.5, CD4-Alexa Fluor 700, CD25-PE, CD25-PE-Cy7, Foxp3-FITC, Foxp3- Percp-Cy5.5, Foxp3-BV421, CD127-APC, CD39-APC, LAP-PE, IL-10-PE, TIM-3-PE, TIM-3-BV421, IFN-γ receptor-PE, IFN-γ- APC, Granzyme B-PE-dazzle E, PD-1-APC, PD-1- PE-Cy7, PD-1- Percp-Cy5.5, CTLA-4-PE, CTLA-4-FITC, ki67-Alexa Fluor 488 and their respective isotype controls were purchased from Biolegend. Recombinant human IFN-γ (R&D Systems) was used at 200 ng/ml. Anti-PD-1 Ab (Nivolumab from Bristol-Myers Squibb), anti-Tim-3 (clone 2E2 from Biolegend) and isotype were used at 10µg/ml.

Liu Z., McMichael E.L., Shayan G., Li J., Chen K., Srivastava R., Kane L.P., Lu B, & Ferris R.L. (2018). Novel effector phenotype of Tim-3+ regulatory T cells leads to enhanced suppressive function in head and neck cancer patients. Clinical cancer research : an official journal of the American Association for Cancer Research, 24(18), 4529-4538.

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Hematopoietic Stem Cell Reconstitution

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Reconstitution assays were performed by transplanting single cell suspensions of BoyJ (CD45.1) or Ubc-GFP⁺ whole bone marrow (WBM) from donor mice into recipients preconditioned with a dose of non-myeloablative radiation (260, 525 or 735 rads) or into recipients preconditioned with a myeloablative dose of radiation (1050 rads). Recipients were either non-tamoxifen treated congenic Wt or R4^KO mice or tamoxifen treated: controls or R4^cKO hosts. Recipient mice were bled at the indicated intervals post-transplantation via the tail vein for analysis of donor chimerism in peripheral blood detectable by GFP or CD45.1 expression. RBC lysed blood was stained with B220-APCy7, CD3-PE or CD3-Alexa Fluor 700, Mac1-PECy7, Ter119-PECy5, Gr1-Pacific Blue, and CD61-APC (Biolegend) to detect mature lineage subtypes for both host and donor mice. Donor bone marrow HSC (Lin^-/cKit⁺/Sca1⁺/Flk2^-/CD45.1⁺ or Lin^-/cKit⁺/Sca1⁺/Flk2^-/GFP⁺) populations were stained as previously described [16 (link),17 (link),24 (link)–28 (link)].

Smith-Berdan S., Bercasio A., Kramer L., Petkus B., Hinck L, & Forsberg E.C. (2021). Acute and endothelial-specific Robo4 deletion affect hematopoietic stem cell trafficking independent of VCAM1. PLoS ONE, 16(8), e0255606.

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Multiparametric Flow Cytometry Profiling of T Cell Subsets

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The anti-mouse TCRβ (mTCR)- PerCp-Cy5.5, CD8a-eFluor 450, were purchased from eBioscience. CD3-APC-Cy7, CD4- PE/FITC/APC, 4–1BB- APC/PE, CD8- PE-Cy7/FITC, OX40- FITC/PE-Cy7, CD62L-PE, and CD45RO-APC/BV421 were purchased from BD (Becton Dickinson Biosciences). CD3-Alexa Fluor 700, CD62L-PE-Cy7, and the live/dead stains- DAPI/PI were purchased from Biolegend.
For FACS sample preparation, cells were harvested and washed with FACS buffer. Cells were resuspended in FACS buffer at a concentration of 1e6–50e6 cells/ml and extracellular fluorescent-conjugated primary antibodies were added and mixed in. After a 20–60 min incubation at 4^oC, which was also protected from light exposure, the samples’ cells were washed and resuspended with FACS buffer.
The flow cytometry assays were performed on FACSCanto I/II (BD Biosciences) and the acquired data were analyzed with FlowJo software (TreeStar). Cells were sorted to: Live/CD3⁺, separated for CD4⁺ or CD8⁺, T_EMRA/ T_EM/T_CM based on sorting out CD62L⁺/CD45RO^- (T naïve cells). Cocultures were sorted for enrichment or into single reactive cells based on 41BB⁺/OX40⁺ (both double and single-positive cells), Live/CD3⁺, separated for CD4⁺ or CD8⁺ by SH800S/MA900 instrument (Sony Biotechnology) or by FACSAria II (BD).

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Comprehensive Immune Phenotyping of PBMCs

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PBMC were thawed and stained with the LIVE/DEAD Aqua (Life Technologies, Grand Island, NY) viability dye according to the manufacturer protocol. Before fixation, cells were incubated with Human Fc Receptor Binding Inhibitor (eBioscience, San Diego, CA) then stained with the following monoclonal antibodies: CD38-PE-TexasRed (Life Technologies), IgD-FITC (BD Biosciences, San Jose, CA), IgM-Brilliant Violet 421, CD307d (FcRL4)-PE, CD3-Alexa Fluor 700, CD10-PE/Cy7, Streptavidin-APC/Cy7, CD19-PerCP/C5.5 (BioLegend, San Diego, CA), CD24-Biotin, CD21-APC, and CD27-650NC (eBioscience). Cells were then fixed in 2% formaldehyde and analyzed within 1-2 hours on an LSR Fortessa (BD Biosciences). An average of 8×10⁵ events were collected (range of 2×10⁵ to 2×10⁶ events), which resulted in an average of 6.7×10⁴ total B cells analyzed (range of 5×10³ to 2×10⁵ B cells). All flow cytometry data was processed using FlowJo Software (Tree Star Inc., San Carlos, CA). Percentages presented for total CD19+ B cell population (CD19+, CD3−) are derived from the percentage of cells within the live gate based on LIVE/DEAD Aqua staining that is within the lymphocyte gate. All B cell subset percentages are the frequency of the total B cell gate (CD19+, CD3−) described above, with the exception of the IgM+ memory B cell subset, which is presented as frequency of CD19+, CD3−, CD10−, CD27+, and IgD−.

Wilmore J.R., Asito A.S., Wei C., Piriou E., Sumba P.O., Sanz I, & Rochford R. (2014). AID expression in peripheral blood of children living in a malaria endemic region is associated with changes in B cell subsets and Epstein-Barr virus. International journal of cancer. Journal international du cancer, 136(6), 1371-1380.

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Multiparameter Flow Cytometry Profiling of Immune Cells

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In study 1, 100 μL fresh EDTA blood was stained directly with a pre-prepared and antibody mixture containing: CD3-AlexaFluor700 (Biolegend; clone OKT3), pan-γδTCR-PE (Beckman Coulter; clone IMMU510), CD56-Brilliant Violet(BV)421 (Biolegend; clone HCD56), CD16-APC-eFluor780 (eBiosciences; clone CB16), CD69-PerCP-Cy5.5 (Biolegend; clone FN50), NKp30-APC (Biolegend; clone P30-15), NKG2D-Brilliant Violet(BV)510 (Biolegend; clone 1D11), NKG2A-PEVio770 (Miltenyi Biotec; clone REA110), and CD57-FITC (Biolegend; clone HCD57). A single mixture was prepared one day before the first time point, aliquotted per time point and stored in the dark until use. Samples were stained at 4°C in the dark for 30 min, followed by erythrocyte lysis with 1 mL FACS Lysis buffer (BD Biosciences) for exactly 5 min. Samples were centrifuged and then washed with 0.5% Bovine Serum Albumin (BSA) in PBS. Cell pellets were resuspended in 100 μL 1% paraformaldehyde (PFA) and analyzed on a Gallios flow cytometer (Beckman Coulter). At each time point, staining and fixation was completed within 4 h of blood draw and flow cytometry was performed the same day using identical acquisition settings and a standardized protocol. CD69 was used as a marker for lymphocyte activation after CHMI, as described earlier (20 (link), 21 (link)).

Walk J, & Sauerwein R.W. (2019). Activatory Receptor NKp30 Predicts NK Cell Activation During Controlled Human Malaria Infection. Frontiers in Immunology, 10, 2864.

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