To determine the tumor CD8+ T lymphocyte infiltration, tumors embedded in OCT were sectioned at 10 μm using a cryostat (Leica Microsystems), and the sections were stained with antibodies directed against the murine CD3 and CD8. After permeabilization with 0.2% (v/v) Triton X-100 (Sigma) in PBS and blocking in 10% (w/v) goat serum, 5% rat serum, and 2% BSA in PBS for 1 hr at room temperature, the primary antibodies (rat CD8a-allophycocyanin [APC] 1:250 [clone 53-6.7, BD Biosciences] and CD3e-fluorescein isothiocyanate (FITC) 1:1,000 [clone 145-2C11, BD Biosciences]) were applied to the slides for 1 hr at room temperature. After being washed with PBS, the sections were mounted using Vectashield Mounting Medium (Vector Laboratories) containing DAPI to visualize the cell nuclei. The slides were imaged using a structured illumination AxioImager microscope (Zeiss, 10× objective).
Cd3e fluorescein isothiocyanate fitc
Manufactured by BD
CD3e-fluorescein isothiocyanate (FITC) is a fluorescent-labeled antibody that binds to the CD3e subunit of the T-cell receptor complex. It is used in flow cytometry applications to identify and quantify T-cells in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Axioimager microscope, by Zeiss (1 mentions)
Clone 53 6, by BD (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
Cryostat, by Leica (1 mentions)
Cd31 pe, by BD (1 mentions)
Cd11c pe cy7, by BD (1 mentions)
Cd25 pe cy7, by BD (1 mentions)
Monensin, by Thermo Fisher Scientific (1 mentions)
Cd80 percp efluor710, by BD (1 mentions)
B7 h4 pe, by BD (1 mentions)
Cd83 fitc, by BD (1 mentions)
Cd107a pe, by BD (1 mentions)
Cd8a pe cy7, by BD (1 mentions)
Perforin fitc, by BD (1 mentions)
Il 4 pe cy7, by BD (1 mentions)
Ifn γ alexa fluor 488, by BD (1 mentions)
Foxp3 pe, by BD (1 mentions)
Phorbol 12 myristate 13 acetate, by Merck Group (1 mentions)
Lsrfortessa, by BD (1 mentions)
2 protocols using cd3e fluorescein isothiocyanate fitc
1
Quantifying Tumor CD8+ T Cell Infiltration
2
Flow Cytometry Immunophenotyping Protocol
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Cell phenotype was assessed by flow cytometry (BD LSRFortessa; BD Biosciences) after incubation with the following fluorescein-labeled antibodies: CD45-allophycocyanin (APC)-eFluor780, CD3e-fluorescein isothiocyanate (FITC), CD4-APC, CD8a-phycoerythrin (PE)-cy7, CD25-PE-cy7, Foxp3-PE, IFN-γ-AlexaFluor488, IL-4-PE-cy7, CD107a-PE, granzymeB-peridinin chlorophyll (PerCP)-eFluor710, perforin-FITC, CD11c-PE-cy7, CD80-PerCP-eFluor710, CD83-FITC, B7-H4-PE, and CD31-PE. All antibodies were purchased from eBioscience (San Diego, CA, USA), except CD4-APC, CD8a-PE-cy7, and Foxp3-PE (BD Biosciences). Intracellular antigens were determined after incubation with ionomycin (500 ng/mL; Abcam, Cambridge, UK) and phorbol-12-myristate13-acetate (10 ng/mL; Sigma-Aldrich, St. Louis, MO, USA) for 1 h and monensin (2 μM; eBioscience) for an additional 4 h. Fixation and permeabilization were performed prior to antibody incubation, and data were analyzed using FlowJo software (TreeStar, Ashland, OR, USA).
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