Cd8 fitc

Manufactured by Beckman Coulter

Sourced in United States

The CD8-FITC is a fluorescently labeled monoclonal antibody that binds to the CD8 antigen expressed on the surface of certain T cells. It is used for the identification and enumeration of CD8+ T cells in flow cytometry analysis.

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Close spelling variants of this product

Anti-CD8-FITC (14 citations) Cyto-STAT tetraCHROME CD45-FITC/CD4-RD1/CD8-ECD/CD3-PC5 (5 citations) CD3-PC5/CD4-FITC/CD8-PE (4 citations) Anti-CD8-FITC (clone T8, (2 citations) CD4-FITC/CD8-FITC (2 citations) FITC-conjugated anti-CD8 (2 citations) CD45-FITC/CD4-PE/CD8-ECD/CD3-PC5 (2 citations)

7 protocols using cd8 fitc

Phenotypic Characterization of Cell Subsets

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Various subpopulations within the cell products (PBMCs prior to culture and cultured DC-CIKs) were identified by flow cytometry, as previously described (11 (link)), using the following fluorochrome-conjugated antibodies: CD3 PerCP-Cy5.5, CD4 FITC, CD8 FITC, CD25 PE, CD28 PE, CD56 PE (all Beckman Coulter, Inc.), PD-1 PE, lymphocyte-activation gene 3 (LAG-3) PE, tumor necrosis factor receptor superfamily member 9 (4-1BB; CD137) PE and T cell immunoglobulin and mucin protein 3 (TIM-3) PeCy-7 (all BioLegend, Inc.). Three-color flow cytometry analysis was performed on a Cytomics FC500 flow cytometer with CXP analysis software (Beckman Coulter, Inc.).

Huang L., Qiao G., Morse M.A., Wang X., Zhou X., Wu J., Hobeika A., Ren J, & Lyerly H.K. (2019). Predictive significance of T cell subset changes during ex vivo generation of adoptive cellular therapy products for the treatment of advanced non-small cell lung cancer. Oncology Letters, 18(6), 5717-5724.

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Immunophenotyping of Whole Blood Cells

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Whole blood samples (200 µL) were incubated with conjugated antibodies as indicated in the dark for 10 minutes at room temperature before red blood cells were lysed. Samples pellets were re-suspended in 500 µL phosphate-buffered saline (PBS) after centrifugation for 5 minutes at 1,300 g at room temperature and analyzed by flow cytometry.
Conjugated antibodies used in this study were purchased from Beckman Coulter (Brea, CA, USA) and included CD3-PC5/CD4-FITC/CD8-PE (IM1650), CD3-FITC/(CD16⁺/CD56)-PE (A07735), CD(14+16)-FITC/CD85k(ILT3)-PE/CD33-PC5 (A23413), CD4-FITC (A007750), CD8-FITC (A07756), CD19-PC5 (A07771), and CD25-PE (A07774).
Beckman-Coulter FC500 (Beckman Coulter, USA) and CXP analysis software (Beckman Coulter) was used for flow cytometry. There are 10,000 gated events in every analysis. Lymphocytes subtypes were selected according to physical characteristics including volume and transmissivity. The level of T lymphocyte subtype was expressed as percentage of the total number of lymphocytes.

Shao B., Li H., Liu X., Song G., Jiang H., Yan Y., Zhang R., Ran R., Zhang J., Liu Y., Wang H., Wang J, & Di L. (2023). The prognostic value of neutrophil-to-lymphocyte ratio in de novo stage IV breast cancer: a retrospective cohort study. Annals of Translational Medicine, 11(2), 45.

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T Cell Proliferation and Checkpoint Receptor Analysis

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T cells were labeled with 1 M CFSE (Invitrogen, Grand Island, NY), cultured with CD3/CD28 beads (Invitrogen, Grand Island, NY) for 3 days, stained with antibodies against CD4 or CD8 (Beckman Coulter, Indianapolis, IN) and fixed for flow cytometric analysis on a FACS Calibur (BD Biosciences). Percent proliferation was determined based on dilution of CFSE in CD4+ or CD8+ cells. For analysis of T cell activation checkpoint receptor expression, T cells were stained with CD4-APC, CD8-FITC (Beckman Coulter) and PD-1 or CTLA-4 antibodies (BD Biosciences, San Jose, CA) after 3 days of culture with CD3/CD28 beads. For analysis of apoptosis, cells were stained with APC-Annexin V (BD Biosciences, San Jose, CA) and propidium iodide (BD Biosciences, San Jose, CA) as recommended by the manufacturer.

Mace T.A., King S.A., Ameen Z., Elnaggar O., Young G., Riedl K.M., Schwartz S.J., Clinton S.K., Knobloch T.J., Weghorst C.M, & Lesinski G.B. (2014). Bioactive compounds or metabolites from black raspberries modulate T lymphocyte proliferation, myeloid cell differentiation and Jak/STAT signaling. Cancer immunology, immunotherapy : CII, 63(9), 889-900.

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Peripheral Blood Mononuclear Cell Immunophenotyping

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Peripheral blood mononuclear cells were resuspended in 0.5% Human Serum Albumin in PBS and 200 ng/ml purified mouse IgG (Sigma) prior to incubation with CD8-FITC (Beckman-Coulter) and either phycoerythrin E75, E90, or negative tetramer (iTAg^™ MHC Class I Human Tetramer-SA-PE, (MBL International Corporation, Woburn MA). Stained cells were washed and fixed in 1% formalin (Polysciences) in PBS an hour before acquisition on FACSCaliber^™ flow cytometer (BD Biosciences).

, & Stern M.E. (1999). Separation of a midlevel density current from the bottom of a continental slope. Proceedings of the National Academy of Sciences of the United States of America, 96(4), 1206-1211.

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Beckman-Coulter Antibody Panel Analysis

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The Beckman-Coulter company supplied the following antibodies: CD4 FITC (RPT-T4), CD19 FITC (HIB19), CD3 PC-5 (UCHT1), CD8 FITC (RTP-T8), HLA-A2 FITC (BB7.2), and MS IGG1 KPA ITCL FITC (MOPC-21).

Zhong H., Xibing G., Yaping D., Zheng W., Decai F., Xiaoye G., Hangyuan W., Dong W, & Zhonghua L. (2016). Interleukin-7 in Patients With Chronic Hepatitis B May Have Effect on T Follicular Helper Cells and Specific Cellular Immunity. Hepatitis Monthly, 16(9), e36068.

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Multiparameter Flow Cytometry Analysis

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Peripheral blood samples (200 μL) were incubated with anti-human antibody cocktails in the dark for 10 min at room temperature, and samples were then subjected to hemolysis for an additional 10 min. The cells were centrifuged for 5 min at 1300× g at room temperature and resuspended in 500 μL phosphate-buffered saline for flow cytometry. Antibody cocktails included CD3-PC5/CD4-FITC/CD8-PE (IM1650), CD3-FITC/CD16/CD56-PE (A07735), CD14/16-FITC/CD85k (ILT3)-PE/CD33-PC5 (A23413), CD4-FITC (A007750), CD8-FITC (A07756), CD19-PC5 (A07771), and CD25-PE (A07774) (all from Beckman Coulter, Brea, CA, USA). Flow cytometry was performed using an FC500 and CXP analysis software (both Beckman Coulter). Each analysis included 10,000 gated events.

Shao B., Liu X., Li H., Song G., Di L., Jiang H., Yan Y., Zhang R., Ran R., Zhang J., Liu Y., Wang H, & Wang J. (2022). Prognostic Value of Pretreatment Neutrophil-to-Lymphocyte Ratio in HER2-Positive Metastatic Breast Cancer. Current Oncology, 29(9), 6154-6166.

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In Vitro Blood-Brain Barrier Migration

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Transmigration was assessed using the hCMEC/D3 cell line as an in vitro model of human blood-brain barrier (Merck Millipore). The day before migration experiments, 3 × 10⁵ endothelial cells were seeded on a collagen-coated 3 μm pore size transwell (Falcon; Corning, Corning, NY) in EndoGRO medium (Merck Millipore, Burlington, MA) as previously described.^{21 (link)} PBMCs were stained with the following antihuman antibodies: CD4-PE, CD8-FITC (Beckman Coulter), and CD3-APC/Fire 750 (Biolegend, San Diego, CA). CD3⁺CD4⁺CD8⁻ T cells were sorted using an FACS Aria II (BD Biosciences). Then, 5 × 10⁵ CD4⁺ T cells in complete Roswell Park Memorial Institute medium supplemented with 10% of fetal bovine serum were added in the upper chamber of a transwell. After 20 hours at 37°C, migrated and nonmigrated cells were collected. For both compartments, cells were stained as described earlier and analyzed by flow cytometry (FACS Celesta; BD Biosciences).

Morille J., Mandon M., Rodriguez S., Roulois D., Leonard S., Garcia A., Wiertlewski S., Le Page E., Berthelot L., Nicot A., Mathé C., Lejeune F., Tarte K., Delaloy C., Amé P., Laplaud D, & Michel L. (2022). Multiple Sclerosis CSF Is Enriched With Follicular T Cells Displaying a Th1/Eomes Signature. Neurology® Neuroimmunology & Neuroinflammation, 9(6), e200033.

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