Thymic stromal cells were isolated as described previously [41 (link), 78 (link)]. Briefly, thymi were minced with razor blades and digested with DNase I and Liberase TM (Roche) before gradient centrifugation with Percoll PLUS (GE Healthcare). Enriched stromal cells were first incubated with the Fc-receptor blocking antibody 2.4G2 and then stained with the indicated surface marker antibodies (BioLegend). For lymphocyte staining, all surface marker antibodies were obtained from BioLegend. For intracellular staining, cells were processed using the Foxp3 Staining Buffer Set and stained with anti-Foxp3-APC (eBiosciences), anti-Ki67-PE (BD Biosciences), or anti-Aire-A647 (eBiosciences). All data were collected using a BD LSR II flow cytometer and analyzed with either FloJo software (TreeStar) or FACS Diva (BD Biosciences). Cell sorting for microarray and qPCR analyses was performed using a BD FACS Aria III cell sorter.
Anti ki67 pe
Manufactured by BD
Sourced in United States
The Anti-Ki67-PE is a fluorescent-labeled antibody for detecting the presence of the Ki67 protein, which is a marker of cellular proliferation. It can be used in flow cytometry and microscopy applications to measure the expression of Ki67 in cell samples.
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Lab products found in correlation
Anti foxp3 apc, by Thermo Fisher Scientific (3 mentions)
Foxp3 staining buffer set, by Thermo Fisher Scientific (2 mentions)
Facsdiva, by BD (2 mentions)
Liberase, by Roche (2 mentions)
Dnase 1, by Roche (2 mentions)
Anti aire a647, by Thermo Fisher Scientific (2 mentions)
Percoll plus, by GE Healthcare (2 mentions)
Facsdiva software, by BD (2 mentions)
Anti cd4 percp, by BD (2 mentions)
Facsaria 3 cell sorter, by BD (1 mentions)
Surface marker antibodies, by BioLegend (1 mentions)
Flojo software, by Tree Star (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Anti cd45ra fitc, by BD (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Facsaria cytometer, by BD (1 mentions)
Ficoll paque plus, by Cytiva (1 mentions)
Anti helios pe, by Thermo Fisher Scientific (1 mentions)
Anti ccr4 pe, by BD (1 mentions)
Anti tnfr2 pe, by R&D Systems (1 mentions)
10 protocols using anti ki67 pe
1
Isolation and Characterization of Thymic Stromal Cells
2
Phenotypic Analysis of PBMC and Liver Cells
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PBMCs were isolated from heparinized blood samples by Ficoll-Paque plus (Amersham, Uppsala, Sweden) density gradient. After isolation, cells were washed two times with RPMI-1640 (Gibco, Auckland, N.Z.) and prepared for further study. The surface and intracellular stainings were performed using the following fluorochrome-conjugated antibodies: anti-CD4-PerCP, anti-CD45RA-FITC, anti-CCR4-PE, anti-CCR5-PE, anti-CCR7-PE, anti-Ki67-PE (BD Pharmingen, San Diego, CA), anti-Tim3-PE, anti-TNFR2-PE (R&D Systems, Minneapolis, MN), anti-Helios-PE, anti-FoxP3-APC (eBioscience, San Diego, CA). For intracellular staining, cells were fixed and permeabilized using the Human FoxP3 Buffer Set (eBiosciences, San Diego, CA) according to the manufacturer’s instructions. Isotype-matched control antibodies were used to define the positive staining populations. Stained cells were acquired and analyzed using a FACSAria cytometer (Becton Dickinson, San Jose, CA, USA) with FACSDiVa software (BD Biosciences).
Liver biopsy specimen was obtained from six chronic HBV infected patients and was chopped and incubated with collagenase-D (0.1%) (Gibco, Waltham, USA) in RPMI-1640 with 10% fetal bovine serum (Gibco, Grand Island, NY). After incubate and filtered through nylon mesh, cells were suspended in RPMI-1640 and then stained with anti-CD4-PerCP, anti-CD45RA-FITC, anti-Tim3-PE and anti-FoxP3-APC.
Liver biopsy specimen was obtained from six chronic HBV infected patients and was chopped and incubated with collagenase-D (0.1%) (Gibco, Waltham, USA) in RPMI-1640 with 10% fetal bovine serum (Gibco, Grand Island, NY). After incubate and filtered through nylon mesh, cells were suspended in RPMI-1640 and then stained with anti-CD4-PerCP, anti-CD45RA-FITC, anti-Tim3-PE and anti-FoxP3-APC.
3
Phenotyping and Proliferation of Anti-SHIV T Cells
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Surface markers and intracellular Ki67were stained for phenotyping and measuring in vivo adoptively transferred and proliferating anti-SHIV T Cells. After PBMCs were isolated from the blood and tissue samples, cells were washed twice with PBS and stained with LIVE/DEAD® Fixable Aqua Dead Cell Stain Kit (Thermo Fisher SCIENTIFIC) for 20 min at room temperature followed by staining with the following antibodies: CD3-Cy7-APC (Clone: SP34.2, BD Biosciences), CD4-BV605 (Clone: OKT4, BioLegend), CD8-BV711 (Clone: RPA-T8, BioLegend), CD28-Cy5PE (Clone: 28.2, BD Biosciences), CD45RA-Cy7-PE (Clone: L48, BD Pharmingen), CD69-ECD (Clone: TP1.55.3, Beckman Coulter), CCR7-Alexa Flour 680 (Clone: 150503, BD Pharmingen) and HLA-DR-BV421 (Clone: LN3, Biolegend). After surface staining, cells were permeabilized with Foxp3 / Transcription Factor Staining Buffer Set (eBiosciences) for 20 min at room temperature, then intracellular stained with anti-Ki67-PE (Clone: B56, BD Pharmingen).
4
Isolation and Analysis of Thymic Stromal Cells
5
Multiparametric Immunophenotyping of Cultured Cells
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For marker analysis, the cells were detached using Accutase (Thermo Fisher Scientific) and washed by adding PBS supplemented with 2 mM EDTA and 0.5% BSA. Cells were centrifuged at 300 g and 4°C, incubated for 15 min in Cytofix fixation buffer (Becton Dickinson), washed with PBS, and centrifuged under aforementioned condition. The pellets were re-suspended in an adequate volume of staining buffer comprised of PBS supplemented with 0.5% BSA and 2 mM EDTA. Cell concentration, antibody dilution, and incubation conditions were used as recommended in the antibody datasheet. The fluorochrome-conjugated antibodies were Anti-CD90-PE, Anti-CD105-APC, Anti-CD73-PE, Anti-CD271/p75-APC, Anti-HNK-1-FITC, Anti-E-CADHERIN/CDH1-APC, Anti-GFAP-A647, Anti-PSA-NCAM-APC, Anti-MAP2-A647, Anti-NESTIN-PE, Anti-IgG1-FITC, Anti-IgG1-PE, Anti-IgG1-APC (all from Miltenyi Biotec), DAPI (BD Biosciences), and Anti-Ki67-PE (BD Biosciences). Stained cells were washed by centrifugation at 300×g and re-suspended in 500 μL PBS supplemented with 2 mM EDTA. Sample acquisitions and analysis were done with an Attune™ flow cytometer (Thermo Fisher Scientific) and FlowJo software, respectively.
6
Comprehensive Immune Cell Profiling
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The following mAbs were used for flow cytometry: anti-CD3 PerCP, anti-CD4 FITC, anti-CD4 PerCP, anti-CD4 PE-Cy7, anti-CD8 PErCP, anti-CD8 APC-Cy7, anti-CD28 FITC, anti-CD95 APC, anti-CD195 (anti-CCR5) PE, anti-Ki67 PE, anti-HLA-DR APC, and anti-HLA-DR FITC (BD Biosciences). Additionally, annexin V PE was purchased from BD Biosciences, anti-CD8 APC and anti-CD8 PE were obtained from Beckman Coulter (CA, USA), and anti-CD38 FITC was purchased from StemCell Technologies (Vancouver, Canada). Flow cytometry data were acquired on a FACSAria II (BD Biosciences). All data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA).
7
Flow Cytometric Apoptosis and Proliferation Assay
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For flow cytometric analysis, detached cells were resuspended in PBS and washed twice by centrifugation. FITC Annexin V Apoptosis Detection Kit I (BD Biosciences, Franklin Lakes, NJ, USA) was used to detect apoptotic and dead cells. The cells were stained with FITC Annexin V and Propidium Iodide (PI) for 15 min according to the manufacturer’s instructions.
Prior to intracellular staining, fixation and permeabilization of cells were performed using BD Cytofix/Cytoperm™ solution (BD Bioscience, Franklin Lakes, NJ, USA). One-half million permeabilized cells were stained with anti-Ki67-PE (BD Bioscience, Franklin Lakes, NJ, USA) or anti-Ki67-BV420 (Sony Biotechnology, Tokyo, Japan) monoclonal antibody. Unstained or treated with non-specific isotypic antibodies conjugated with PE samples served as negative controls. All samples were washed twice with Perm/Wash™ buffer (BD Bioscience, Franklin Lakes, NJ, USA) containing saponin. The FACSAria III flow cytometer/cell sorter (BD Bioscience, Franklin Lakes, NJ, USA) was used for the viability and Ki67 expression analyses.
Prior to intracellular staining, fixation and permeabilization of cells were performed using BD Cytofix/Cytoperm™ solution (BD Bioscience, Franklin Lakes, NJ, USA). One-half million permeabilized cells were stained with anti-Ki67-PE (BD Bioscience, Franklin Lakes, NJ, USA) or anti-Ki67-BV420 (Sony Biotechnology, Tokyo, Japan) monoclonal antibody. Unstained or treated with non-specific isotypic antibodies conjugated with PE samples served as negative controls. All samples were washed twice with Perm/Wash™ buffer (BD Bioscience, Franklin Lakes, NJ, USA) containing saponin. The FACSAria III flow cytometer/cell sorter (BD Bioscience, Franklin Lakes, NJ, USA) was used for the viability and Ki67 expression analyses.
8
Immune Cell Profiling in Testes
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Immune cells in peripheral blood were collected and counts and phenotypes were analyzed by flow cytometry. The original data are reported in our previous studies (33 (link), 34 (link)). Interstitial cells in the testes were isolated as described previously (10 (link)). Briefly, testicular tissues were chopped and then incubated at 37°C with collagenase and DNase for 1 h. Tubule fragments were allowed to settle for 3 min, followed by interstitial cell recovery in PBS. Subsequently, the phenotypes of the interstitial cells were analyzed by multi-parameter flow cytometry. The anti-human flow cytometry antibodies cross-reactive with NPMs included anti-CD45-PE (557059, BD, USA), anti-CD3-APC-Cy7 (557757, BD), anti-CD20-FITC (302304, BioLegend, USA), anti-CD4-PerCP-Cy5.5 (317428, BioLegend), anti-CD8-PE-Cy7 (557746, BD), anti-Ki67-PE (51-36525X, BD), and anti-PD-1-PE (329906, BioLegend).
9
Tumor Dissociation and Flow Cytometry Analysis
10
Single-Cell Analysis of Melanoma Signaling
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For analysis of single cells, flow cytometry was used. All collected cells were fixed for 15 min in 1.6% paraformaldehyde (PFA) at room temperature (RT) and permeabilized with 100% ice-cold methanol. Four samples (representing non-treated and differently treated samples) were barcoded with different concentrations of Pacific Orange or Pacific Blue dye (both ThermoFisher Scientific, Waltham, MA), as described previously [19] and illustrated in Supplementary Figure S1. Following incubation at RT for 30 min and a washing step, the four barcoded samples were combined into one tube and stained simultaneously with different antibodies for 30 min at RT: anti-pS6-Alexa Fluor 647 (#4851) 1:200 and anti-p-c-Jun (#3270) 1:200 (both from Cell Signaling Technology, Danvers, MA); anti-Ki67-PE (#556027) 1:6 and anti-fibronectin-Alexa Fluor 647 (#563098) 1:40 (both from BD Pharmingen, San Jose, CA); anti-Melan-A (M7196) 1:66 (DakoCytomation, Glostrup, Denmark). The stained samples were analyzed on a LSR II flow cytometer controlled by BD FACS Diva TM software (BD Bioscience, San Jose, CA). The data was analyzed by using Flow Jo software (FlowJo, Ashland, OR). The melanoma cells were distinguished from the fibroblasts based on the GFP signal, and only the GFP + cancer cells were analyzed for the levels of proteins of interest, as illustrated in Supplementary Figure S1.
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