Mouse anti-mouse PD-1 monoclonal antibody was a kind gift from Dr. Gordon Freeman (Dana-Farber Cancer Institute, Harvard University, Boston, MA, USA). Rat anti-mouse PD-1 monoclonal antibody (Catalog# BP0146; Clone RMP1-14) was purchased from BioXCell Inc. (West Lebanon, NH, USA). Purified and Fluorochrome-conjugated antibodies (TruStain FcX anti-mouse CD16/32, Catalog#101319; anti-mouse CD80-FITC, catalog#104705; anti-mouse CD44-FITC, catalog#103005; anti-mouse CD24-PE, catalog#138503; anti-mouseCD47-PE, catalog# 127507; anti-mouse IL-12-PE, catalog#505203; anti-mouse CD8-APC, catalog#100712; anti-mouse Gr1-APC, catalog#108411; anti-mouse PD-L1-APC, catalog#124311; and anti-mouse CD4-BV711, catalog#100447) for flow cytometry analysis were purchased from BioLegend Inc. (San Diego, CA, USA). FITC-conjugated secondary antibodies (goat anti-mouse IgG-FITC, catalog# 115-095062) were purchased from Jackson Immunoresearch Labs (Bar Harbor, ME, USA).
Apc anti mouse cd8
Manufactured by BioLegend
Sourced in United States
APC anti-mouse CD8 is a fluorophore-conjugated antibody that binds to the CD8 receptor expressed on the surface of certain T cells in mice. It can be used to identify and quantify CD8-positive cells in flow cytometry applications.
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Lab products found in correlation
Pe anti mouse cd4, by BioLegend (4 mentions)
Fitc anti mouse cd11b, by BioLegend (2 mentions)
Anti mouse cd3 percp, by BioLegend (2 mentions)
Anti mouse ly6c apc, by BioLegend (2 mentions)
Pe anti mouse ly6g, by BioLegend (2 mentions)
Fitc anti mouse cd3, by BioLegend (2 mentions)
Fitc anti mouse cd80, by BioLegend (1 mentions)
Goat anti mouse igg fitc, by Jackson ImmunoResearch (1 mentions)
Trustain fcx anti mouse cd16 32, by BioLegend (1 mentions)
Anti mouse gr1 apc, by BioLegend (1 mentions)
Anti mouse cd4 bv711, by BioLegend (1 mentions)
Antimouse cd3 percp cyanine 5, by BioLegend (1 mentions)
Anti cd8a ly 2 microbeads, by Miltenyi Biotec (1 mentions)
Golgiplug, by BD (1 mentions)
Fortessa flow cytometer, by BD (1 mentions)
Anti mouse il 2 pe, by BioLegend (1 mentions)
Anti mouse ifn γ pe cy7, by BioLegend (1 mentions)
Pe anti mouse cd25, by BioLegend (1 mentions)
Pe anti human cd8, by BioLegend (1 mentions)
Anti pd 1 pe, by BioLegend (1 mentions)
15 protocols using apc anti mouse cd8
1
Multicolor Flow Cytometry Panel
2
Isolation of MDSCs and CD8+ T Cells from Tumor-Bearing Mice
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Male BALB/c mice aged 4–5 weeks were inoculated with CT26 cells to obtain tumor‐bearing mice according to the protocol of animal model. Two weeks after CT26 cells inoculation, mice were sacrificed and spleens were collected to filter through a 70 µm filter to prepare a single cell suspension. Then, mononuclear cells were obtained using a lymphocyte separation medium. MDSCs were isolated using the antimouse Gr‐1‐Biotin antibody and antibiotin MicroBeads (Miltenyi Biotech, Germany) according to the protocol of the MDSC‐kit. The purity of isolated MDSCs was identified by staining with antimouse CD11b‐FITC (BioLegend, USA) and antimouse Gr‐1‐PE/Cy7 (BioLegend, USA) antibodies and further detected by flow cytometry. For CD8+ T cells purification, normal BALB/c mice aged 4–5 weeks were sacrificed and spleens were collected to filter through a 70 µm filter to prepare a single cell suspension. Similarly, mononuclear cells were obtained using lymphocyte separation medium and CD8+ T cells were directly isolated using the anti‐CD8a (Ly‐2) MicroBeads (Miltenyi Biotech, Germany) according to the protocol. The purity of isolated CD8+ T cells was identified by incubating with antibodies of antimouse CD3‐PerCP/Cyanine 5.5 (BioLegend, USA) and antimouse CD8‐APC (BioLegend, USA) and measured by flow cytometry analysis.
3
ZIKV, DENV, and HIV Peptide-Specific Immune Responses
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For flow cytometric (FACS) analysis, 3×106 freshly isolated mouse splenocytes were seeded in 24-well plate and then stimulated with either 10μg/ml E.coli-ZIKV-E80, or 10μg/ml S2-ZIKV-E80, or 10μg/ml DENV-E80, or 10μg/ml HIV peptide, or 50ng/ml PMA and 500ng/ml ionomycin as the positive control, and PBS as a negative control. Golgi plug (BD Biosciences) was added two hours after stimulation at the dilution of 1:1000. After an additional 4 hours, splenocytes were collected and stained with the following antibodies: anti-mouse CD3 (Alexa fluro® 488, Biolegend), anti-mouse CD4 (BV786, BD Bioscience), anti-mouse CD8 (APC, Biolegend), anti-mouse IL-2 (PE, Biolegend), anti-mouse IFN-γ (PE/Cy7, Biolegend), and Aqua fluorescent for live and dead cell discrimination (Life technologies). The stained cells were fixed in 4% paraformaldehyde. FACS analysis was performed on a Fortessa flow cytometer (BD Biosciences), and data were analyzed with FlowJo software (Tree Star).
4
Multiparameter Flow Cytometry Analysis
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Primary antibodies used for this study includes Annexin V-FITC (Biolegend Cat# 640906), anti-PD-1-PE (BioLegend Cat# 621607), anti-human CD8-PE (BioLegend Cat# 344706), anti-mouse CD4-PE (BioLegend Cat# 100408), anti-mouse CD25-PE (BioLegend Cat# 102012), anti-mouse FOXP3-Alexa Fluor® 488 (BioLegend Cat# 126405), anti-mouse CD8-APC (BioLegend Cat# 100711), anti-mouse PD-1-PE (BioLegend Cat# 135205), IgG isotype control-PE (BioLegend Cat# 400907), anti-human CD4-APC (eBioscience Cat# RPA-T4). FoxP3 staining used FoxP3 buffer (BioLegend Cat#421403). Samples were analyzed using FACSCalibur and FlowJo software.
5
T Cell Subpopulations Analysis in Mouse Spleen
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The percentages of T cell subpopulations in the spleens of mice in the experimental groups were detected by flow cytometry. The spleens were obtained 2 weeks after the final immunization from mice (n = 6) in each group, and a flow cytometry assay was performed as previously described (11 (link)). Briefly, 1 × 106 splenocytes were suspended in pre-cooled PBS and incubated with anti-mouse CD3-PerCP, anti-mouse CD4-PE, and anti-mouse CD8-APC antibodies (all from BioLegend) at 4°C for 30 min in the dark. Then, the cells were washed twice with pre-cooled PBS, resuspended in PBS and analyzed with a FACSAria flow cytometer (BD Biosciences), with 20,000 total events/sample. Data were analyzed by FlowJo software (Tree Star Inc.).
6
Murine Spleen T Cell Subpopulations
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Flow cytometry was used to analyse the percentages of T cell subpopulations in spleens of mice in the experimental groups. The spleens were obtained two weeks after the nal immunization from mice (n=6) in each group, and a ow cytometry assay was performed as previously described [10] . Brie y, 1×10 6 splenocytes were suspended in 100 μl pre-cooled PBS and incubated with 0.5 μg anti-mouse CD3-PerCP, 0.25 μg anti-mouse CD4-PE and 0.25 μg anti-mouse CD8-APC (all from BioLegend) at 4 °C for 30 min in the dark. Then, the cells were washed twice with pre-cooled PBS, resuspended in PBS and analysed with a FACSAria ow cytometer (BD Biosciences) with 20,000 total events/sample. Data were analysed by FlowJo software (Tree Star Inc.).
7
Comprehensive Murine Immune Cell Profiling
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The following antibodies were used for FACS staining: APC/Cy7 anti-mouse CD3 (100221, Biolegend, San Diego, CA, USA), FITC anti-mouse CD4 (553729, BD Bioscience, San Jose, CA, USA), FITC anti-mouse CD8 (553031, BD Bioscience), APC anti-mouse CD8 (100711, Biolegend), APC anti-mouse Foxp3 (4331294, Invitrogen, Waltham, MA, USA), APC anti-mouse CD62L (104411, Biolegend), PE anti-mouse CD44 (553134, BD Bioscience), APC anti-mouse CD49b (108909, Biolegend), FITC anti-mouse CD11b (557396, BD Bioscience), H-2Kb/OVA (SIINFEKL)-Tetramer/PE was kindly provided by Prof. Eui-Cheol Shin (KAIST, Daejeon, Korea).
8
Multiparametric Flow Cytometry of Myeloid-Derived Suppressor Cells
9
Multiparameter Flow Cytometry Immunophenotyping
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Blood was drawn via cardiac puncture and incubated with ammonium-chloride-potassium lysing buffer for red blood cell lysis. Blood leukocytes were subsequently blocked with anti-mouse CD16/32 (14–0161–81; eBioscience) and stained with fluorophore-conjugated antibodies A700 anti-mouse CD45 (103127; BioLegend), PE anti-mouse CD11b (101208; BioLegend), APC-Cy7 anti-mouse CD11c (117323; BioLegend), PE-Cy7 anti-mouse CD4 (100421; BioLegend), APC anti-mouse CD8 (100711; BioLegend) and FITC anti-mouse CD19 (115505; BioLegend) as well as propidium iodide (PI; Invitrogen) for live/dead discrimination. Data were acquired on a BD FACSAria II and analyzed using the FlowJo software to assess peripheral cell counts. Gating was performed following routinely used protocols, using FSC/SSC to exclude dead cells, cell debris and doublets and propidium iodide to identify dead cells. At least 250,000 events were captured by the cytometer and 30,000 single cells analyzed per group. Cells were gated on CD45+PI− live singlets. Dead cells were excluded as propidium iodide (PI)-positive. Populations of live singlets were then gated was percentages of CD45 + PI- cells.
10
Quantifying Tumor Cell Surface PD-L1 Expression
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To measure PDL1 levels on the cell surface of tumour cells treated with vehicle, cisplatin, radiation, JQ1, IFN‐γ (20 ng/ml,24 PeproTech), or the combinations for the different time periods were trypsinized and harvested for staining with streptavidin‐phycoerythrin (PE)‐conjugated anti‐human PD‐L1(329706, Biolegend). Tumour tissues were collected, cut into small pieces, digested with a mixture of collagenase, DNase and hyaluronidase for preparing single‐cell suspension and then treated with Cell Stimulation Cocktail (00‐4975‐03, eBioscience) as instructed. Cells were washed, re‐suspended in fluorescence‐activated cell sorting buffer at 4°C and then stained with fluorescent‐conjugated antibodies and appropriate isotype controls for multicolor flow cytometry. For cell surface staining, the following antibodies were used: fluorescein isothiocyanate (FITC) anti‐mouse CD3 (100204, Biolegend), PE/Cy7 anti‐mouse cluster of differentiation 4 (CD4) (100422, Biolegend), APC anti‐mouse CD8 (100712, Biolegend), Brilliant Violet 605 anti‐mouse NK1.1 (108739, Biolegend) and PE anti‐mouse PD‐L1(124308, Biolegend). The collected live single cells were fixed and treated with permeabilization buffer before intracellular staining with Brilliant Violet 421 anti‐mouse IFN‐γ (505830, Biolegend).
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