Tumors were minced and digested in a 1 mg/mL collagenase type IA (Gibco, Gaithersburg, MD) solution for 1 h at 37 °C. Cells were filtered through a 100 μm nylon mesh cell strainer (BD Falcon, Bedford, MA) and stained with fluorophore-conjugated antibodies for the identification and quantitative analysis of Tregs, TAMs and MDSCs populations by FACS as previously described13 (link). Labelled cells were analyzed on a FACS Canto II cytometer using FACS Diva 5.5 software (BD Biosciences, San Jose, CA) and analyzed by FlowJo software (Tree Star, San Carlos, CA). The subpopulation of Tregs was identified by FoxP3 and CD45+, CD3+, CD4+, CD8-, CD25+ cell surface markers, while MDSCs were recognized by CD45+, CD11b+ and Gr-1+ markers, and TAMs as CD45+, CD11b+ and F4/80+. Polarization of TAMs towards M2 or M1 phenotype was assessed by measuring CD206 or CD86 surface markers, respectively. In addition, the M2/M1 ratio was computed from the quotient between the mean fluorescence intensity (MFI) values for CD206 and CD86 markers. Isotype controls were employed to establish background fluorescence. Cell populations were measured as cells per gram of tumor and total numbers of TAMs, Tregs and MDSCs recruited within the tumor.
Facs diva 5
Manufactured by BD
The FACS Diva 5.5 software is a flow cytometry analysis and data management tool designed to facilitate the acquisition, analysis, and organization of flow cytometry data. It provides core functionality for the operation and control of flow cytometers.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2 cytometer, by BD (4 mentions)
100 μm nylon mesh cell strainer, by BD (1 mentions)
Collagenase type ia, by Thermo Fisher Scientific (1 mentions)
Ab8219, by Abcam (1 mentions)
A11029, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Merck Group (1 mentions)
Ethylenediaminetetraacetic acid, by Merck Group (1 mentions)
Versene, by Thermo Fisher Scientific (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
Modfit lt software, by Verity Software House (1 mentions)
Cycletest plus dna reagent kit, by BD (1 mentions)
Fitc annexin 5 apoptosis detection kit 1, by BD (1 mentions)
Facs lsrii flow cytometer, by BD (1 mentions)
Fitc mouse anti human cd44 antibody, by BD (1 mentions)
Ab5032, by Merck Group (1 mentions)
8 protocols using facs diva 5
1
Tumor Immune Cell Profiling by FACS
2
CD63 Expression in Lung Cancer Cell Lines
3
Macrophage Phenotype Evaluation under Oxygen
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To determine whether oxygen concentrations could exert a potential effect on tumor cells affecting macrophage polarization, CD86 and CD206 surface markers representative for M1 (anti-tumoral) and M2 (pro-tumoral) macrophage phenotypes, respectively, were appraised using fluorescence activated cell sorting (FACS) analyses. In order to collect RAW264.7 cells remaining on both sides of the transwell membrane, the inserts were firstly rinsed with 1x PBS and then transferred into bottom plates containing 0.05% trypsin/0.02% ethylenediaminetetraacetic acid (Sigma-Aldrich). The obtained cells were stained with CD86-PerCP/Cy5.5 and CD206-APC fluorescence-conjugated antibodies and analyzed on a FACS Canto II cytometer using FACS Diva 5.5 software (BD Biosciences, San Jose, CA). Data were analyzed with FlowJo software (Tree Star, San Carlos, CA) to compute the median fluorescence intensities (MFI) of CD206 and CD86 markers.
4
Aortic Cell Dissociation and Flow Cytometry
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Detailed protocol is provided as Supplementary Information . Dissected aortas were minced and incubated with an aorta dissociation cocktail. For flow cytometry analysis, cells were fixed and stained with specific antibodies. Data were acquired on a FACS CantoII cytometer using the FACS Diva 5.5 software (BD Biosciences, San Jose, CA) and analyzed by FlowJo software (Tree Star, San Carlos, CA). In all of the experiments, macrophages were identified as CD11b-F4/80 double positive cells.
5
Isolation and Sorting of Nestin-GFP+ NLSCs
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Nestin‐GFP+ NLSCs were isolated as mentioned earlier and cultured for 14 days in laminin‐coated dishes containing growth media. Cells were removed from the flask using Versene (Thermo Fisher), counted, spun down at 5,000 rpm for 5 minutes in microfuge tubes, and resuspended in 100 µl 1% PBS‐bovine serum albumin (BSA) per 2 × 105 cells. Aggregates were removed by passing them through a 40‐μm cell strainer prior to sorting. Cells were then incubated on ice for 1 hour and later labeled with 5 μg/ml of rabbit antinerve growth factor receptor (anti‐NGFR) antibody (Advanced Targeting Systems, San Diego, CA, http://atsbio.com ) for 2 hours. After three washings with 1% PBS‐BSA, cells were labeled with Alexa Fluor 647 anti‐rabbit antibody (Thermo Fisher) for 1 hour in the dark on ice. After three washings with 1% PBS‐BSA, cells were resuspended in 1% PBS‐BSA and sorted using a FACS Aria Sorter. Data acquisition and analyses were performed using BD FACS Diva 5.0.3 software.
6
Fluorescence-Activated Cell Sorting Protocol
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FACS was carried out on a BD FACS (Aria Sorter, San Jose, CA) at 4°C and a pressure of 20 psi, using a laser at the 488 nm line, a 530/30 band pass filter, a 100 mm sorting tip, and a 34.2 kHz drive frequency, sterilized with 10% bleach. This instrument allowed us to characterize cells by size as well as fluorescence. Low flow rate improved the purity of cell sorting. Data acquisition and analyses were performed using BD FACS Diva 5.0.3 software, gated for a high level of EGFP expression. The clear separation of EGFP+ from EGFP- cells explains the ease of sorting. Sorted cells were re-analyzed to confirm that all were EGFP+. They were then plated on laminin-coated dishes.
7
Apoptosis and Cell Cycle Analyses
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Apoptosis and cell cycle analyses were performed separately using commercial Annexin V: FITC Apoptosis Detection Kit I and CycleTest™ Plus DNA Reagent kit, respectively (BD Bioscience, San Jose, CA). Cells were seeded at 5 x 105 cells per well in twelve-well plates. After overnight attachment, the cells were treated with the compound at 12.5, 25 and 50 μM for 24 hours for the apoptosis analysis and at 2.5 μM for 12, 24 and 48 hours for the cell cycle analysis. After treatment, the cells were harvested by gentle trypsinization and centrifuged at 1500 x g for 5 minutes. Cell staining was performed according to the manufacturer’s instructions. A DNA QC Particles kit was used to calibrate the FACSCanto II flow cytometer (BD Biosciences, San Jose, CA). Cell populations were subjected to cytometric analysis, and the quadrants were set according to the population of viable cells in the untreated samples. FacsDiva 5.0.3 software (BD Biosciences, San Jose, CA) was used to calculate the percent of cells in the respective quadrants for apoptosis analysis, and Mod Fit LT software (Verity Software House Inc., Topsham, ME) was used for the cell cycle analysis.
8
Quantitative Flow Cytometry Analysis of NCAM and CD44 Expression
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Cells were double labeled with 10 μg/ml of rabbit polyclonal anti-human NCAM antibody (AB5032; Millipore) followed by AlexaFluor 647-labeled goat anti-rabbit secondary antibody (Molecular Probes, Invitrogen, Eugene, OR, USA) and inactive endosialidase-GFP fusion protein [33 (link)] in PBS. Parallel samples were labeled with AlexaFluor 647 mouse anti-human alkaline phosphatase antibody (B4-78; BD Biosciences, San Jose, CA, USA) and FITC mouse anti-human CD44 antibody (BD Biosciences) according to the manufacturer’s instructions. Appropriate fluorescence minus one (FMO) controls were used for analysis. The cells were analyzed with FACS LSR II flow cytometer and FACSDiva 5.0.3 software (BD Biosciences). Cell debris and dead cells were excluded from the analysis based on physical parameters and propidium iodide (PI) fluorescence probing for cell viability (proportion of positive cells 1.2–7.0 %, data not shown).
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