Cd3 alexa700

Manufactured by BioLegend

Sourced in United States

CD3-Alexa700 is a fluorescently-labeled antibody that binds to the CD3 antigen on T cells. It is a tool used in flow cytometry applications to identify and enumerate T cells within a sample.

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Lab products found in correlation

Cd8 apc cy7, by BioLegend (3 mentions) Cd14 apc cy7, by BioLegend (3 mentions) Cd4 alexafluor700, by BD (2 mentions) Cd66b percp cy5.5 g10f5, by BioLegend (2 mentions) Lsr 2 cytometer, by BD (2 mentions) Hla dr bv785, by BioLegend (2 mentions) Cd19 percp cy5, by BioLegend (2 mentions) Ccr7 fitc 150503, by R&D Systems (2 mentions) Cd45ra apc efluor780 hi100, by Thermo Fisher Scientific (2 mentions) Hiv p24 rd1 fh190 1 1, by Beckman Coulter (2 mentions) Cd8 percp cy5, by BioLegend (2 mentions) Cd3 pacific blue, by BD (2 mentions) Foxp3 staining kit, by Thermo Fisher Scientific (2 mentions) Cd4 pacblue, by BioLegend (2 mentions) Cd14 cd20 pacorange, by Thermo Fisher Scientific (2 mentions) Ccr7 pe cy7, by BioLegend (2 mentions) Cd45ra qdot655, by Thermo Fisher Scientific (2 mentions) Cd8 apc cy7, by BD (2 mentions) Cd4 fitc, by BioLegend (2 mentions) Cd133 apc, by Miltenyi Biotec (2 mentions)

12 protocols using cd3 alexa700

Multiparametric Flow Cytometry Immunophenotyping

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The BLCs and PBMCs were counted and assessed for viability using trypan blue (Sigma-Aldrich) and compound microscopy to ensure >90% lymphocyte viability. To assess distribution of CD4⁺ and CD8⁺ T cells, immune regulation and functionality of CD8⁺ T cells in HIV infection, cells from the two compartments were subjected to two panels of fluorescently labeled antibodies. Panel 1 (phenotypic panel): Live/dead-amcyan (Life Technologies, Carlsbad, CA, USA), CD3-BV650, CD4-BV711, CD14-APC-Cy7, PD-1-BV711, TIM-3-BV785 (BioLegend, San Diego, CA, USA), and CD8-PE Texas Red (Invitrogen, Carlsbad, CA, USA). Panel 2 (intracellular cytokine staining panel): Live/dead-amcyan (Life Technologies), CD3-Alexa700, CD4-BV711, CD8-APC-Cy7, granzyme b-Alexa647, IFNγ-Dazzle 549 (BioLegend). Panel 2 staining was done after stimulation with PMA/Ionomycin (25/500 ng/mL). Acquisition was performed on BD FACS Aria III. Flowjo v10.5 (Flowjo, LLC) was used for flow cytometry analysis.

Muema D.M., Mthembu M., Schiff A.E., Singh U., Corleis B., Chen D., Bassett T., Rasehlo S.S., Nyamande K., Khan D.F., Maharaj P., Mitha M., Suleman M., Mhlane Z., Naidoo T., Ramjit D., Karim F., Kwon D.S., Ndung'u T, & Wong E.B. (2020). Contrasting Inflammatory Signatures in Peripheral Blood and Bronchoalveolar Cells Reveal Compartment-Specific Effects of HIV Infection. Frontiers in Immunology, 11, 864.

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Multicolor Flow Cytometry Panel

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Fluorphore-conjugated monoclonal antibodies from BD Bioscience (CD3-V500, CD8-V500, CD3-Alexa 700 and TNF-α-PE-Cy7), Biolegend (CD14-FITC, CD16-FITC, CD19-FITC, CD14-PerCP/Cy5.5, CD16-PerCP, CD19-PerCP, IFN-γ-Pacific Blue, PD-1-PerCP/Cy5.5, IL-2-APC and CD4-Alexa 700) and eBioscience (CD127-V450) and Invitrogen (LIVE/DEAD blue fluorescent reactive dye and CD8-Qdot 605) were used in these studies.

Callendret B., Eccleston H.B., Satterfield W., Capone S., Folgori A., Cortese R., Nicosia A, & Walker C.M. (2015). Persistent HCV replication despite priming of functional CD8+ T cells by combined therapy with a vaccine and direct acting antiviral. Hepatology (Baltimore, Md.), 63(5), 1442-1454.

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Polychromatic Flow Cytometry Analysis

Cited 1 time

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The following fluorochrome-conjugated Abs were used for polychromatic flow cytometry analysis: CD3-Pacific-blue (UCHT1), CD4-Alexa-Fluor 700 (RPA-T4), CCR4-PE-Cy7 (1G1), CCR6-PE (11A9), CCR7-PE-Cy7 (3D12), CXCR3-PE-Cy5 (1C6) (BD Biosciences, San Diego, CA), CCR7-FITC (150503) (R&D Systems, Minneapolis, MN), CD45RA-APC-eFluor780 (HI100) (eBioscience, San Diego, CA), CD3-Alexa700 (UCHT1), CD326-BV650 (9C4), CD8-PerCP-Cy5.5 (RPA-T8), CD19-PerCP-Cy5.5 (HIB19), CD66b-PerCP-Cy5.5 (G10F5), HLADR-BV785 (L243; BioLegend), CD4-FITC (SFCI12T4D11) and HIV-p24-RD1 (FH190-1-1; Beckman Coulter, Fullerton, CA). Live/Dead Fixable Aqua Dead Cell Stain Kit (Vivid; Life Technologies, Burlington, ON) was used to exclude dead cells. Cells were analyzed by FACS using the BD-LSRII cytometer and BD-Diva (BD Biosciences, San Jose, CA), and FlowJo (^©Tree Star, Inc., Ashland, OR) softwares. All Abs were titrated for an optimal noise/signal ratio and Ab cocktails were validated by comparing single to multiple staining. Positive gates were placed based on fluorescence minus one (FMO) controls [41 (link), 69 ]

Gosselin A., Wiche Salinas T.R., Planas D., Wacleche V.S., Zhang Y., Fromentin R., Chomont N., Cohen É.A., Shacklett B., Mehraj V., Ghali M.P., Routy J.P, & Ancuta P. (2016). HIV persists in CCR6+CD4+ T cells from colon and blood during antiretroviral therapy. AIDS (London, England), 31(1), 35-48.

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Phenotyping Peripheral Blood Immune Cells

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Standard extracellular staining was performed on PBMCs using the following fluorophore-labeled antibodies: CD3-Alexa700 (Biolegend), CD14/CD20-PacOrange (Invitrogen), CD8-APC-Cy7 (BD pharmingen), CD4-Pac Blue (Biolegend), CD45RA-Qdot655 (Invitrogen), CCR7-PECy7 (Biolegend), CD25-PE (Biolegend). For FoxP3 staining, cells were stained using the FoxP3 staining kit (eBioscience) according to manufacturer’s protocol. Frequencies of cell populations and co-signalling molecule expression were determined by flow cytometry.

Diller M.L., Kudchadkar R.R., Delman K.A., Lawson D.H, & Ford M.L. (2016). Exogenous IL-2 induces FoxP3+ Th17 cells in vivo in melanoma patients. Journal of immunotherapy (Hagerstown, Md. : 1997), 39(9), 355-366.

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Comprehensive Murine and Human Immune Profiling

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Flow cytometry antibodies for murine samples included CD3e-PE, PD1-BV605, CD45-BV786 (BDbiosciences), CD90.1-FITC, CD62L-PECy7, CD45.1-PE (ebioscience), CD45.2-Alexa700, CD-8α-Percp Cy5.5, CD4-FITC, CD103-APC, CD24-APCcy7 (Invitrogen), CD44-APCy7, CD4-BV421, F4/80-PercpCy5.5, CD39-PECy7, PDL1-PE, CD90.2-Alexa 700, MHCII-BV421, CD11b-BV650, Ly6C-BV711 (Biolegend), CD8α-PE-Texas red (life technologies), CD278 (ICOS)-PE (Biolegend). Flow cytometry antibodies for human samples included CD45-BV510, CD3-Alexa700, CD4-BV785, CD8-BV711, CTLA-4-PE/Dazzle 594, 4-1BB-PE (Biolegend), CD39-BV650, PD-1-PE-Cy7 and Ki-67 Alexa 488 (BD Biosciences), CD103-APC and FOXP3-Alexa 700 (eBioscience).
Mouse antibodies for the explant assays included anti-PD1 (RMP1-14) and anti-CTLA-4 (9D9) by BioXCell and OX40 (OX86) kindly provided by Dr. Andrew Weinberg (EACRI). Human antibodies for the explant assays included anti-PD1 and anti-CTLA4 purchased from Invitrogen and anti-OX40 kindly provided by Dr. Andrew Weinberg (EACRI).

Sharon S., Duhen T., Bambina S., Baird J., Leidner R., Bell B., Casap N., Crittenden M., Vasudevan S., Jubran M., Kravchenko-Balasha N, & Gough M. (2021). Explant Modeling of the Immune Environment of Head and Neck Cancer. Frontiers in Oncology, 11, 611365.

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Vasculogenic Potential of MNC-QQ

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Release tests for MNC-QQ were conducted to evaluate potency based on the ratio of cells and their vasculogenic potential. The presence of cell types essential for vascular and tissue regeneration in the MNC-QQ culture was confirmed using flow cytometry antibody-based detection of the following cell surface markers: CD34-PE, CD206-PE/Cy7, CD192(CCR2)-PerCP/Cy5.5, CD3-Alexa700, CD14-APC/Cy7, CD4-FITC, CD8-APC/Cy7, CD31-APC/Cy7, CD19-FITC, CD56-BV421, CD25-PerCP/Cy5.5, CD127-BV421 (BioLegend, San Diego, CA, USA), CD133-APC (Miltenyi Biotec, San Diego, CA, USA), and CXCR4-APC (BD Biosciences).^{17 (link),23 (link)}The vasculogenic potential of MNC-QQ was evaluated using a MethoCult SF H4236 colony formation assay (STEMCELL Technologies, Vancouver, BC, Canada).^{22 (link)} Cells (2 × 10⁵ per dish) were inoculated into a 35-mm Primaria culture dish and cultured for 14 days under routine conditions. Clusters comprising more than 100 cells were counted as primitive EPC CFU or definitive CFU based on the cell size and shape under phase-contrast microscopy (Nikon, Tokyo, Japan). PB collected less than 1 month before cell therapy was used to compare the baseline vasculogenic potential and cell surface markers between PBMNCs and MNC-QQ cells harvested on the treatment date.

Tanaka R., Fujimura S., Kado M., Fukuta T., Arita K., Hirano-Ito R., Mita T., Watada H., Kato Y., Miyauchi K, & Mizuno H. (2022). Phase I/IIa Feasibility Trial of Autologous Quality- and Quantity-Cultured Peripheral Blood Mononuclear Cell Therapy for Non-Healing Extremity Ulcers. Stem Cells Translational Medicine, 11(2), 146-158.

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Polychromatic Flow Cytometry of Immune Markers

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The following fluorochrome-conjugated antibodies (Abs) were used for polychromatic flow cytometry analysis: CD3-Pacific-blue (UCHT1), CD4-Alexa-Fluor 700 (RPA-T4), CCR4-PE-Cy7 (1G1), CCR6-PE (11A9), CCR7-PE-Cy7 (3D12), CXCR3-PE-Cy5 (1C6) (BD Biosciences, San Diego, California, USA), CCR7-FITC (150503) (R&D Systems, Minneapolis, Minnesota, USA), CD45RA-APC-eFluor780 (HI100) (eBioscience, San Diego, California, USA), CD3-Alexa700 (UCHT1), CD326-BV650 (9C4), CD8-PerCP-Cy5.5 (RPA-T8), CD19-PerCP-Cy5.5 (HIB19), CD66b-PerCP-Cy5.5 (G10F5), human leukocyte antigen - antigen D related (HLA-DR)-BV785 (L243; Biolegend ,San Diego, California USA), CD4-FITC (SFCI12T4D11), and HIV-p24-RD1 (FH190–1–1); (Beckman Coulter, Fullerton, California, USA). Live/Dead Fixable Aqua Dead Cell Stain Kit (Vivid; Life Technologies, Burlington, Ontario, Canada) was used to exclude dead cells. Cells were analyzed by fluorescence-activated cell sorting (FACS) using the BD-LSRII cytometer and BD-Diva (BD Biosciences) and FlowJo (Tree Star, Inc., Ashland, Oregon, USA) softwares. All Abs were titrated for an optimal noise/signal ratio and Ab cocktails were validated by comparing single with multiple staining. Positive gates were placed based on fluorescence minus one controls [41 (link),69 ].

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Phenotypic analysis of PBMC subsets

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Extracellular flow cytometry staining was performed on PBMCs using the following fluorophore-labeled antibodies: CD3-Alexa700 (Biolegend), CD14/CD20-PacOrange (Invitrogen), CD8-APC-Cy7 (BD pharmingen), CD4-Pac Blue (Biolegend), CD45RA-Qdot655 (Invitrogen), CCR7-PECy7 (Biolegend). For FoxP3 staining, cells were stained using the FoxP3 staining kit (eBioscience) according to manufacturer’s protocol. Frequencies of cell populations and co-signalling molecule expression were determined by flow cytometry. The flow cytometer was calibrated daily using Cytometer Setup and Tracking (CS&T) beads (BD pharmingen). Mid-range beads were utilized and photomultiplier tube (PMT) voltages adjusted prior to analyzing each experiment. Data were analyzed using FlowJo software (TreeStar, San Carlos, CA).

Diller M.L., Kudchadkar R.R., Delman K.A., Lawson D.H, & Ford M.L. (2016). Complete response to high dose IL-2 (HDIL-2) and enhanced IFNγ+Th17:TREG ratio in a melanoma patient. Melanoma research, 26(5), 535-539.

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Sorting and Profiling Plasmablasts

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Staining for sorting was performed using cryo-preserved PBMCs in 2% FBS and 2 mM ethylenediaminetetraacetic acid (EDTA) in PBS (P2). Cells were stained for 30 min on ice with CD20-Pacific Blue (2H7, 1:400), Zombie Aqua, CD71-FITC (CY1G4, 1:200), IgD-PerCP-Cy5.5 (IA6–2, 1:200), CD19-PE (HIB19, 1:200), CD38-PE-Cy7 (HIT2, 1:200), and CD3-Alexa 700 (HIT3a, 1:200), all BioLegend. Cells were washed twice, and single plasmablasts (live singlet CD19⁺ CD3⁻ IgD^lo CD38⁺ CD20⁻ CD71⁺) were sorted using a FACSAria II into 96-well plates containing 2 μL Lysis Buffer (Clontech) supplemented with 1 U/μL RNase inhibitor (NEB) and immediately frozen on dry ice, or bulk sorted into PBS supplemented with 0.05% BSA and processed for single cell RNAseq.

Amanat F., Thapa M., Lei T., Sayed Ahmed S.M., Adelsberg D.C., Carreno J.M., Strohmeier S., Schmitz A.J., Zafar S., Zhou J.Q., Rijnink W., Alshammary H., Borcherding N., Reiche A.G., Srivastava K., Sordillo E.M., van Bakel H., Turner J.S., Bajic G., Simon V., Ellebedy A.H, & Krammer F. (2021). The plasmablast response to SARS-CoV-2 mRNA vaccination is dominated by non-neutralizing antibodies and targets both the NTD and the RBD. medRxiv.

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Multiparametric Immunophenotyping of HCV-specific T Cells

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Surface and intracellular staining was performed according to standard protocols on previously frozen PBMCs using monoclonal antibodies (CD3–PE-TexRed (BeckmanCoulter, Brea, CA), CD3-Alexa-700, CD4-PacificBlue, CD8-APC-Cy7, HLA-DR-PE-Cy7, CD38–PerCP-Cy5.5, TNFα-APC, IFNγ-APC (Biolegend, San Diego, CA), Ki-67-FITC, Bcl-2-PE and LIVE/DEAD Stain Kit (Invitrogen, Frederick, MD). BD FACS Permeabilizing Solution (BectonDickinson) was used for intracellular staining. For detection of HCV-specific proliferating cells PBMCs were stimulated with peptide pools (5 ug/mL as for ELIspot) specific for NS3, NS5A and NS5B. Brefeldin (10ug/mL) (Sigma, St.Louis, MO) was added after 18 hours to arrest cytokine release and incubation was continued for a total of 24 hours before surface and intracellular staining. Data for FACS analyses were collected with LSR II and analyzed with FlowJo software 9.1.

Zubkova I., Duan H., Wells F., Mostowski H., Chang E., Pirollo K., Krawczynski K., Lanford R, & Major M. (2014). Hepatitis C virus clearance correlates with HLA-DR expression on proliferating CD8+ T-cells in immune-primed chimpanzees. Hepatology (Baltimore, Md.), 59(3), 803-813.

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