For T cell proliferation assay, splenocytes derived from naïve C57BL/6 mice were labelled with 5 μM Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) (Biolegend) and incubated for 10 minutes at 37°C in the dark. Cells were then resuspended in RPMI 1640 supplemented with 10% FBS and stimulated with Dynabeads Mouse T-Activator CD3/CD28. Activated splenocytes (S) were then co-cultured with bone marrow derived macrophages (BMMs) from WT and Grn-/- mice or with macrophages (M) magnetically isolated cells from day 6 and day 14 metastasis bearing livers (4:1 ratio, S: M). Cells were plated in 96 well plates and incubated at 37°C for 72 hours. Subsequently, cells were harvested and stained with CD8 antibody (Biolegend, clone 53-6.7). Proliferating CD8+ T cells were tracked by flow cytometry. For some experiments, recombinant mouse Progranulin protein (R&D systems; [1 µg/ml]) and mouse Periostin neutralizing antibody (R&D systems, [1 µg/ml]) were used.
Cd8 antibody
Manufactured by BioLegend
Sourced in United States
The CD8 antibody is a laboratory reagent used for the detection and analysis of CD8+ T cells in various research applications. It is a protein that specifically binds to the CD8 surface molecule expressed on cytotoxic T lymphocytes. The CD8 antibody can be used in flow cytometry, immunohistochemistry, and other immunological techniques to identify and quantify CD8+ T cells in biological samples.
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Lab products found in correlation
Cd45ro antibody, by BioLegend (2 mentions)
Ccr7 antibody, by BioLegend (2 mentions)
Lsrfortessa system, by BD (2 mentions)
Mouse periostin neutralizing antibody, by R&D Systems (1 mentions)
Cm1950, by Leica (1 mentions)
Bx41 microscope, by Olympus (1 mentions)
Fc block cd16 cd32, by BD (1 mentions)
Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Fluorophore conjugated antibody, by BioLegend (1 mentions)
Facsaria iiiu, by BD (1 mentions)
Ic fixation buffer, by Thermo Fisher Scientific (1 mentions)
Brefeldin a, by Thermo Fisher Scientific (1 mentions)
Permeabilization buffer, by Thermo Fisher Scientific (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Phorbol 12 myristate 13 acetate, by Merck Group (1 mentions)
Facscanto 2, by BD (1 mentions)
96 well round bottom plate, by Corning (1 mentions)
Goat f ab 2 anti human igg pe, by Southern Biotech (1 mentions)
Facscelesta, by BD (1 mentions)
Lsrii ow cytometer, by BD (1 mentions)
7 protocols using cd8 antibody
1
Analyzing T Cell Proliferation in Tumor Microenvironment
2
Quantifying CAR T Cell Phenotypes
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One million CAR T cells were stained with primary antibodies for antigen–antibody binding reaction. After 30 min of incubation in the dark, unbound antibodies and solutes were washed with a PBS rinse. This method was used to detect T cell transfection rates, with the following primary antibodies used: CD4 antibody (BioLegend Cat# 357408, USA), CD8 antibody (BioLegend Cat# 344714), CD45RO antibody (BioLegend Cat# 304206), CCR7 antibody (BioLegend Cat# 353214). Target cells were harvested and resuspended in cold PBS containing 2% FBS. The anti-MUC16 antibody was added to the cell suspension for incubation for 1 h. The cells were centrifuged, resuspended, and incubated with Cy3-labeled secondary antibodies in PBS containing 2% FBS for 30 min prior to two washes with PBS. For all flow cytometry experiments, appropriate IgG isotype controls were used to assess nonspecific staining. The specificity of MUC16 antibodies was confirmed by utilizing control IgG matched to the species. All flow cytometry data were collected using a BD LSRFortessa system (BD Biosciences, USA) and analyzed with FlowJo software (FlowJo, Oregon, USA). All flow antibodies are listed in Additional file 8 : Table S3.
3
Immunohistochemical Analysis of T-cell Subsets
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The tissues were collected at different time points, dipped in isopentane and frozen at −80°C. Tissues were cut into 6 μm sections by freezing microtome (Leica, CM1950). The sections were fixed with −20°C acetone for 10 min, washed three times with PBS. Then the slides were incubated with 3% H2O2 for 8 min, washed twice with PBS and slides blocked with 10% goat serum for 30 min. Slides were incubated with the primary antibodies at 4°C overnight diluted in PBS. Following incubation, the slides were rinsed 3 times with PBS. Polink-2 plus polymer HRP detection system for rat primary antibody (ZSGB-BIO, PV-9004) and DAB kit (ZSGB-BIO, ZLI-9018) were used. The slides were flushed using tap water thoroughly, dyed by hematoxylin, flushed by tap water, differentiated by hydrochloric acid alcohol, dehydrated, and sealed. The images were visualized using an Olympus BX41 microscope. Dilutions and catalog numbers for primary antibodies were as follows: CD4 antibody (1:100 dilution) (BioLegend, 100402), CD8 antibody (1:100 dilution) (BioLegend, 100702).
Ten high-power fields (HPF) were randomly selected from each group to count lymphocytes, three mice per group at each time point. Statistical graphs show the mean ± standard error of the mean (SEM) of each group and compared using independent T-test.
Ten high-power fields (HPF) were randomly selected from each group to count lymphocytes, three mice per group at each time point. Statistical graphs show the mean ± standard error of the mean (SEM) of each group and compared using independent T-test.
4
Multiparameter Flow Cytometry Analysis of Immune Cells
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Cells (1 × 106) were collected, washed twice with 1× phosphate-buffered saline (PBS), and resuspended in 1 mL 1× PBS containing 2% FBS. Then, labeled primary antibodies were used to stain cells following the manufacturer’s instruction for 1 hour at 4°C in the dark followed by washing twice and detection by flow cytometry. The following primary antibodies were used: CD7 antibody (BioLegend catalogue# 343104; CA), CD4 antibody (BioLegend catalogue# 357408), CD8 antibody (BioLegend catalogue# 344714), CD45RO antibody (BioLegend catalogue# 304206), CCR7 antibody (BioLegend catalogue# 353214), programmed cell death protein 1 antibody (BioLegend catalogue#329932), and mucin domain containing 3 antibody (BioLegend catalogue#345008). All flow cytometry data were obtained with a BD LSRFortessa system (BD Biosciences) and analyzed with FlowJo software (FlowJo, OR).
5
Isolation and Analysis of Metastatic Liver CD8+ T Cells
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Single-cell suspensions from murine livers were prepared as outlined above, followed by resuspension in MAC buffer (0.5% BSA, 2 mM EDTA, PBS). Cells were blocked for 10 min on ice with FC block (CD16/CD32, BD Biosciences), and then stained with Sytox-blue viability marker (Thermo Fisher) and fluorophore-conjugated antibodies (Biolegend), listed in Supplementary Table 1 .
To assess IFNγ expression levels in metastasis-derived CD8+ T cells, magnetically isolated CD8a+ T cells from metastatic livers were stimulated with 50 ng/mL phorbol 12-myristate 13-acetate (Sigma Aldrich) and 1 μg/mL of ionomycin (Sigma Aldrich) for 5 h at 37 °C in the presence of brefeldin A (eBioscience; 1:100). For intracellular staining, cells were first fixed (eBioscience, IC fixation buffer) and permeabilized (eBioscience, 1× permeabilization buffer). Cells were stained with fluorophore-conjugated IFNγ antibody (Biolegend, clone XMG1.2), CD8 antibody (biolegend) and LIVE/DEAD™ fixable Aqua Dead Cell Stain Kit (Thermo Fisher).
Flow cytometry was performed on a FACS Canto II (BD Biosciences), and fluorescence- activated cell sorting (FACS) was carried out using FACS Aria IIIu (BD Biosciences). Cells were sorted directly into RLT buffer + β-mercaptoethanol according to the manufacturers instruction for RNA isolation (Qiagen).
To assess IFNγ expression levels in metastasis-derived CD8+ T cells, magnetically isolated CD8a+ T cells from metastatic livers were stimulated with 50 ng/mL phorbol 12-myristate 13-acetate (Sigma Aldrich) and 1 μg/mL of ionomycin (Sigma Aldrich) for 5 h at 37 °C in the presence of brefeldin A (eBioscience; 1:100). For intracellular staining, cells were first fixed (eBioscience, IC fixation buffer) and permeabilized (eBioscience, 1× permeabilization buffer). Cells were stained with fluorophore-conjugated IFNγ antibody (Biolegend, clone XMG1.2), CD8 antibody (biolegend) and LIVE/DEAD™ fixable Aqua Dead Cell Stain Kit (Thermo Fisher).
Flow cytometry was performed on a FACS Canto II (BD Biosciences), and fluorescence- activated cell sorting (FACS) was carried out using FACS Aria IIIu (BD Biosciences). Cells were sorted directly into RLT buffer + β-mercaptoethanol according to the manufacturers instruction for RNA isolation (Qiagen).
6
Flow Cytometry Antibody Screening Protocol
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All washes and dilutions of cells, antibodies, and reagents were performed using flow buffer (1X PBS, 1% BSA, 0.1% NaN3, pH 7.4). Staining was performed in a round-bottom 96-well plate (Corning) seeded at 100,000 cells/well and all incubations were performed at 4 °C or on ice. For primary and secondary screens, the cells were incubated for 30 min with pre-diluted test antibodies (secondary screen and dose-curves) or 1:5 diluted HEK 293 supernatants containing antibodies (for primary screens and diversity screens) in a total volume of 50 μL. The cells were washed twice with 200 µL flow buffer. The cells were then incubated for 30 min with detection antibody (Goat F(ab')2 Anti-Human IgG-PE, Southern Biotech) at 0.625 µg/mL in flow buffer. Following 2 more washes, the cells were resuspended in a final volume of 150 µL of flow buffer. The cells were analyzed on a BD FACSCelesta or a Guava easyCyte 8-HT flow cytometer. At least 3000 events were collected, and PE geometric mean fluorescence intensity was plotted as a fold over background (cells incubated with secondary detection antibody only). In some secondary screens involving human or cynomolgus PBMCs, an additional CD4 antibody (BioLegend) and/or CD8 antibody (BioLegend) was included to further characterize cell binding.
7
CD8+ T Cell Proliferation Assay
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To analyze the CD8 + T cell among CD45 + cells from the of mice, CD8 + T cells were collected and concentrated, and they were incubated with 5 µL of speci c uorescent CD8 antibody (100705, Biolegend, USA) and speci c uorescent CD45 antibody (368507, Biolegend) for 20 min. Saline (500 µL) was added separately into the cell solution, which was followed by centrifugation at 5000 rpm for 5 min.
The supernatant was removed, and the collected cells at the bottom were dispersed into 300 µL of saline.
Finally, the effective separation rate was analyzed by counting 1.2 × 10 4 cells per sample using an LSR II ow cytometer (BD Biosciences, CA, USA) and Cell Quest software (BD Biosciences).
CD8 + T cell proliferation assay CD8 T + cell was puri ed from peripheral blood mononuclear cells (PBMCs) of healthy female volunteers. Cell purity was checked. CD8 + T cellwas labeled with 1μM CFSE (S8269, selleck, USA) and stimulated with CD3/CD28 antibody and co-cultured with vector transfected, LC3plasmid, and LC3 + NLRC5 HEC-1A cells, in 96-well plates at a ratio of 5: 1 for 3 days. CD8 + T cell was collected, and the dilution of intracellular CFSE caused by proliferation was calculated using ow cytometry.
The supernatant was removed, and the collected cells at the bottom were dispersed into 300 µL of saline.
Finally, the effective separation rate was analyzed by counting 1.2 × 10 4 cells per sample using an LSR II ow cytometer (BD Biosciences, CA, USA) and Cell Quest software (BD Biosciences).
CD8 + T cell proliferation assay CD8 T + cell was puri ed from peripheral blood mononuclear cells (PBMCs) of healthy female volunteers. Cell purity was checked. CD8 + T cellwas labeled with 1μM CFSE (S8269, selleck, USA) and stimulated with CD3/CD28 antibody and co-cultured with vector transfected, LC3plasmid, and LC3 + NLRC5 HEC-1A cells, in 96-well plates at a ratio of 5: 1 for 3 days. CD8 + T cell was collected, and the dilution of intracellular CFSE caused by proliferation was calculated using ow cytometry.
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