The Tetramer Facility of National Institutes of Health (NIH) provided LCMV-GP33 tetramer. Cells were stained with allophycocyanin (APC)-labeled GP33 MHC class I tetramer (GP33/H-2Db) for 15 minutes at 37 °C. After incubation, the samples were stained with anti-CD8 (clone 53–6.7; eBioscience) or anti-CD4 (clone GK1.5; eBioscience) antibodies for 30 minutes at 4 °C. Absolute numbers of GP33-specific CD8+ T cells were calculated with fluorescent beads (BD Biosciences) by using fluorescence-activated cell sorting (FACS). For measurement of intracellular IFN-γ, cells were fixed with 2% formaldehyde in PBS for 10 minutes, permeabilized with 1% saponin in FACS buffer at room temperature, and stained with anti–IFN-γ antibody for 30 minutes at 4 °C (clone XMG1.2; eBioscience). All stained cells were analysed with a FACSFortessa (BD Biosciences) flow cytometer, and data were analysed with FlowJo software.
Fluorescent beads
Manufactured by BD
Fluorescent beads are small, spherical particles that emit light when exposed to a light source. They are composed of a polymer matrix that is infused with fluorescent dyes or quantum dots. These beads are designed to be used in various applications, such as flow cytometry, fluorescence microscopy, and biological assays, to track and visualize cells, molecules, or other biological entities.
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Lab products found in correlation
Anti ifn γ antibody, by Thermo Fisher Scientific (2 mentions)
Lysing solution, by BD (2 mentions)
Anti cd8 clone 53 6, by Thermo Fisher Scientific (1 mentions)
Anti cd4 clone gk1, by Thermo Fisher Scientific (1 mentions)
Clone xmg1, by Thermo Fisher Scientific (1 mentions)
Facs fortessa, by BD (1 mentions)
Accuri c6 plus, by BD (1 mentions)
Anti cd8, by BD (1 mentions)
Ifn γ, by Thermo Fisher Scientific (1 mentions)
Maraviroc, by Merck Group (1 mentions)
Classical monocyte isolation kit, by Miltenyi Biotec (1 mentions)
Bx471, by Merck Group (1 mentions)
Control goat igg, by Merck Group (1 mentions)
Matrigel, by BD (1 mentions)
Accuri c6, by BD (1 mentions)
Anti cd8 peridinin chlorophyll protein complex percp, by BD (1 mentions)
Anti cd8 53 6, by BD (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
Lsrfortessa, by BD (1 mentions)
5 protocols using fluorescent beads
1
LCMV-GP33 Tetramer Staining Protocol
2
Cell Quantification by Flow Cytometry
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Cells of the two species are similar in size and morphology, which are difficult to be distinguished and enumerated accurately by an optical microscope. Therefore, flow cytometry (Accuri C6 plus, BD) was employed to analyze cell concentrations every two days. Replicated samples (2 mL) from the different treatment groups were analyzed. For the quantification of cell concentration, an aliquot of a calibrated solution of fluorescent beads (1 μm diameter, BD) was added in each sample as an internal standard. Two species were differentiated by auto-fluorescent signals of chlorophyll and phycocyanin.
3
Quantifying Antigen-Specific CD8+ T Cells
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Tetramers were provided by the National Institutes of Health (NIH) Tetramer Facility (Emory University, Atlanta, GA, USA). Cells were stained with allophycocyanin (APC)-labeled GP-33 major histocompatibility complex class I tetramers (GP-33/H-2Db) for 15 min at 37 °C. After incubation, the samples were stained with anti-CD8 (BD Biosciences, San Diego, NJ, USA) for 30 min at 4 °C. Erythrocytes were then lysed with 1 ml BD lysing solution (BD Biosciences), washed once and analyzed by flow cytometry. Absolute numbers of GP-33-specific CD8+ T cells per microliter of blood were determined by fluorescence-activated cell sorting (FACS) analysis using fluorescent beads (BD Biosciences). IFN-γ was purchased from eBiosciences (San Diego, CA, USA).
4
Monocyte Invasion Assay with DLBCL
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Monocytes were purified by negative selection with a Classical Monocyte Isolation Kit (Miltenyi Biotec), according to the manufacturer’s instructions. Purity exceeded 80%. DLBCL cells were incubated in bottom compartments of 8-µM 24-well Transwell plates (Nunc) at 0.5 × 106 cells per milliliter. Twenty-four hours later, 0.2 × 106 purified monocytes were added to upper compartments. After 2 hours of incubation, cells from the bottom compartment were stained for CD14, and CD14+ monocytes were enumerated by flow cytometry with an Accuri C6 (BD Biosciences) set on a volume-based event acquisition. Flow variations were controlled by the addition of fluorescent beads (BD Biosciences). The invasion assay was performed similarly but the filter was covered with 50 µL of Matrigel (BD Biosciences), and the incubation time was extended to 18 hours. Polyclonal goat anti-CCL5 (R&D Systems) and control goat IgG (Sigma) were used at 10 µg/mL. metCCL5 was used at 100 ng/mL. Maraviroc and BX471 (Sigma) were used at 100 µM and 100 nM, respectively.
5
Quantification of Antigen-specific CD8+ T Cells
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Lymphocytes were stained with anti-CD8 (53-6.7; BD Bioscience). For measurement of intracellular IFN-γ, cells were restimulated with glycoprotein 33 (GP33; Strasbourg, France) for 6 h, fixed with 2% formaldehyde (Sigma-Aldrich; Steinheim, Germany) for 10 min, permeabilized with saponin, and stained with anti-IFN-γ antibody (eBioscience).
Tetramers were provided by the National Institutes of Health (NIH) Tetramer Facility (Emory University, Atlanta, GA, USA). Staining was performed as previously described [40] , Briefly, blood and cells were stained with allophycocyanin (APC)-labeled GP33 MHC class I tetramers (GP33/H-2Db) for 15 minutes at 37°C. After incubation, the samples were stained with anti-CD8 peridinin-chlorophyll-protein complex (PerCP; BD Biosciences) for 30 min at 4°C. Erythrocytes were then lysed with 1 ml BD lysing solution (BD Biosciences), washed once, and analyzed with a flow cytometer LSR Fortessa (BD Biosciences). Absolute numbers of GP33-specific CD8 + T cells were calculated by fluorescence-activated cell sorting (FACS) analysis using fluorescent beads (BD Biosciences).
Tetramers were provided by the National Institutes of Health (NIH) Tetramer Facility (Emory University, Atlanta, GA, USA). Staining was performed as previously described [40] , Briefly, blood and cells were stained with allophycocyanin (APC)-labeled GP33 MHC class I tetramers (GP33/H-2Db) for 15 minutes at 37°C. After incubation, the samples were stained with anti-CD8 peridinin-chlorophyll-protein complex (PerCP; BD Biosciences) for 30 min at 4°C. Erythrocytes were then lysed with 1 ml BD lysing solution (BD Biosciences), washed once, and analyzed with a flow cytometer LSR Fortessa (BD Biosciences). Absolute numbers of GP33-specific CD8 + T cells were calculated by fluorescence-activated cell sorting (FACS) analysis using fluorescent beads (BD Biosciences).
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