Cd3 alexa700

Manufactured by Thermo Fisher Scientific

Sourced in United States

The CD3-Alexa700 is a flow cytometry reagent that detects the CD3 protein, which is a marker for T cells. It is conjugated with the Alexa Fluor 700 fluorescent dye, allowing for detection in the far-red region of the spectrum.

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6 protocols using cd3 alexa700

Multicolor FACS Analysis of Immune Cells

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FACS analyses for different immune cell populations were conducted following standard protocols. Freshly-harvested splenocytes and peripheral blood mononuclear cells (PBMCs) were stained with the immune cell markers listed below and analyzed with BD FACS Diva on LSR II. Antibodies used for FACS analysis: CD3 Alexa700 (eBioscience), CD4 PE-Cy5 (eBioscience), CD8 Alexa488 (eBioscience), CD44 APC (eBioscience), CD62L PE-Cy7 (eBioscience), CD19 PE (eBioscience), CD11b Pe-eFluor 610 (eBioscience), F4/80 PE-Cy5 (eBioscience), Ly-6G(ar-1) APC (eBioscience), CD45 APC-eFluor 780 (eBioscience).

Rangan P., Choi I., Wei M., Navarrete G., Guen E., Brandhorst S., Enyati N., Pasia G., Maesincee D., Ocon V., Abdulridha M, & Longo V.D. (2019). Fasting-Mimicking Diet Modulates Microbiota and Promotes Intestinal Regeneration to Reduce Inflammatory Bowel Disease Pathology. Cell reports, 26(10), 2704-2719.e6.

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Isolation and Phenotyping of Murine Lymphocytes

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The mice were sacrificed by cervical dislocation. The liver and splenic lymphocytes were isolated as done previously with minor modifications (31 (link), 32 (link)). In brief, livers and spleens were homogenized and washed two times with the RPMI-1640 medium (300 × g for 10 min at 4°C). The OptiPrepTM working solution (OPWS)-40% iodixanol (Sigma Chemical, St. Louis, Mo., USA) was added to the liver pellets and then layered with Hank's balanced salt solution (Sigma) while the red blood cell lysing buffer (Sigma R7757) was added to the spleen pellets. The pellets were then washed at least two times with the RMPI-1640 medium (300 × g for 7 min at 4°C) and stained with anti-mouse antibodies, including CD3-Alexa 700, CD4-APC/Cy7 or CD4-FITC, CD8-PE/Cy7, NK1.1-PerCP/Cy5.5, CD25-PE, IFNγ-Alexa Fluor 488, IL4-PE, IL17-Alexa Fluor 647, and FOXP3-APC (all from eBioscience). The staining procedures and analyses were performed according to the manufacturer's protocol. The samples were analyzed by the FACSCalibur flow cytometer (BD Biosciences) as previously described (39 (link)).

Fong F.L., El-Nezami H., Mykkänen O, & Kirjavainen P.V. (2022). The Effects of Single Strains and Mixtures of Probiotic Bacteria on Immune Profile in Liver, Spleen, and Peripheral Blood. Frontiers in Nutrition, 9, 773298.

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Cytokine Secretion by PBMC Assay

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Cytokine secretion by PBMC was assayed by ICS followed by flow cytometry using a previously reported protocol [22 (link)] with some minor changes. Briefly, frozen PBMC were re-stimulated for 18 h in the presence of anti-human CD49d and CD28 (BD Biosciences) and CD107a. Re-stimulation for the final 16 h was carried out in the presence of Brefeldin A (Sigma) and Monensin (Golgi Stop, BD Biosciences). Each sample was re-stimulated with either: 1μg/mL Staphylococcal enterotoxin B (SEB, positive control samples); a single pool of 3D7 AMA1-specific peptides (n=56) at final concentration 2μg/mL each peptide (0.11% total DMSO concentration); and 0.11% DMSO final concentration (un-stimulated peptide control sample). Cells were stained the next day using a Live/Dead marker, as well as for CD4, CD14, CD19, CD8α, CD3, IFN-γ, TNFα, and IL-2. The staining antibodies differed from those previously reported only for CD3 (Alexa 700; clone: UCHTI) and CD19 (eFluor450; clone: HIB19) (eBioscience). Samples were analyzed using a LSRII Flow Cytometer (BD Biosciences) and FlowJo v9.7.5 (Tree Star Inc, USA). Dead cells, monocytes (CD14⁺), and B cells (CD19⁺) were excluded from the analysis (Fig. S4). Background responses in un-stimulated no peptide control cells were subtracted from the antigen-specific peptide responses.

Hodgson S.H., Choudhary P., Elias S.C., Milne K.H., Rampling T.W., Biswas S., Poulton I.D., Miura K., Douglas A.D., Alanine D.G., Illingworth J.J., de Cassan S.C., Zhu D., Nicosia A., Long C.A., Moyle S., Berrie E., Lawrie A.M., Wu Y., Ellis R.D., Hill A.V, & Draper S.J. (2014). Combining Viral Vectored and Protein-in-adjuvant Vaccines Against the Blood-stage Malaria Antigen AMA1: Report on a Phase 1a Clinical Trial. Molecular Therapy, 22(12), 2142-2154.

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Multiparameter Flow Cytometry of Immune Cells

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Single cell suspensions from mouse experiments were stained with Live/dead Aqua (Molecular Probes, Inc.), CD4-PerCP, CD8-Pacific Blue, CD69-BV605, CD62E-PE, CD62E-BV421, CD62P-Alexa647, CD162-AlexaFlour647, HECA454-PE (BD Bioscience), CD25-APC, CD31-BV605, CD105-Pacblue, I/A-I/E-BV421, CD45-APCCy7, EpCAM-APC-Cy7, Podoplanin-PE, CD31-PECy7, EpCAM-FITC, CD64-BV711 (Biolegend), CD11c-PECy5.5 (Invitrogen) CXCR3-PECy7, CD3-Alexa700 and Ly6C-efluor450 (eBioscience). For subsequent detection of CXCL10 mRNA Primeflow^® RNA Assay (eBioscience) was used accordingly to manufacture protocol. Samples were acquired on an LSR-II flow cytometer (BD Biosciences) and analysed using FlowJo software (Tree Star Inc.).
Single cell suspension isolated from human tumors and unaffected colon tissue were stained with Live/dead Aqua (Molecular Probes, Inc.), CD31-Alexa700, (Biolegend), CD4-PerCP, CD8-BV711, CD105-APC, CD14-Alexa700, CD19-APCH7 and CD19-PE-CF594 (BD bioscience) followed by permeabilization with Fix & Perm kit (ADG Bio research GMBH) and staining with CXCL9-FITC (R&D) and CXCL10-PE (Biolegend), flow cytometry analyses were performed as above.

Akeus P., Szeponik L., Ahlmanner F., Sundström P., Alsén S., Gustavsson B., Sparwasser T., Raghavan S, & Quiding-Järbrink M. (2018). Regulatory T cells control endothelial chemokine production and migration of T cells into intestinal tumors of APCmin/+ mice. Cancer Immunology, Immunotherapy, 67(7), 1067-1077.

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Multiparametric Flow Cytometry Analysis of PBMCs

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PBMCs were resuspended in PBS, containing 0.5% w/v BSA and 0.01% sodium azide. PBMCs were incubated with saturating concentrations of fluorescently labeled conjugated mAbs. Analysis of cells was performed using a FACSCanto‐II flowcytometer and FlowJo software (version 9.1 and 10) and for the methods of flow cytometry we adhered to the ‘Guidelines for the use of flow cytometry and cell sorting in immunological studies’ 38. The following mAbs were used for flow cytometry: CD3 Alexa Fluor 700 [557943], CD4 PE‐Cy7 [348809], CD8 PerCP‐Cy5.5 [341050], CD19 Alexa Fluor 700 [557921], CD19 PerCP‐Cy 5.5 [332780], CD20 APC‐H7 [641414], CD20 PerCP‐Cy5.5 [332781], CD25 APC [340907], CD27 APC [337169], CD38 PE [345806], CD38 PE‐Cy7 [335825], HLA‐DR FITC [347400], and IgD PE [555779] from BD (San Jose, USA); CD3 Alexa 700 [56‐0038‐41], CD19 Alexa Fluor 700 [56‐0199‐42], and CD27 APC‐eFluor 780 [47‐0279‐42] from eBioscience (San Diego, USA); and CD27 FITC [M1764] from Sanquin (Amsterdam, the Netherlands). To assess lymphocyte viability TO‐PRO‐3 iodide [T3605] was used (Thermo Fisher Scientific, Massachusetts, USA).

Tuijnenburg P., aan de Kerk D.J., Jansen M.H., Morris B., Lieftink C., Beijersbergen R.L., van Leeuwen E.M, & Kuijpers T.W. (2019). High‐throughput compound screen reveals mTOR inhibitors as potential therapeutics to reduce (auto)antibody production by human plasma cells. European Journal of Immunology, 50(1), 73-85.

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Immune Cell Profiling in Sigmoid Colon

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Sigmoid colon biopsies (SCB) were processed using Liberase DL (Roche Diagnostics). Matched blood was collected and PBMCs)were isolated by gradient density centrifugation. Cells were stained with the following fluorochrome-conjugated antibodies: CD3-Pacific-Blue (UCHT1), CD4-Alexa-Fluor 700 (RPA-T4; BD Biosciences), CD45RA-APC-eFluor780 (HI100) (eBioscience), CD3-Alexa700 (UCHT1), CD326-BV650 (9C4), CD8-PerCP-Cy5.5 (RPA-T8), CD19-PerCP-Cy5.5 (HIB19), CD66b-PerCP-Cy5.5 (G10F5; Biolegend), CD4-FITC (SFCI12T4D11; Beckman Coulter), CD64-APC (10.1.1; Miltenyi). Aqua Vivid Dead Cell Stain Kit (Life Technologies) was used to exclude dead cells. Phenotypic analysis was performed by flow cytometry using BD-LSRII cytometer, BD-Diva (BD Biosciences) and FlowJo software (Tree Star). Detailed sample processing informations are included in our previous publication [70 (link)].

Gabriel E.M., Wiche Salinas T.R., Gosselin A., Larouche-Anctil E., Durand M., Landay A.L., El-Far M., Tremblay C.L., Routy J.P, & Ancuta P. (2021). Overt IL-32 Isoform Expression at Intestinal Level during HIV-1 Infection is Negatively Regulated by IL-17A. AIDS (London, England), 35(12), 1881-1894.

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