Cells were subjected to six-color FACS on a FACSCanto flow cytometer using CellQuest 3.2.1f1 software (Becton Dickinson, San Jose, CA), utilizing monoclonal Abs against CD45, CD3, CD4, CD8, CD14, CD11c, CCR5, CXCR3, Vbeta- 13.1. Data representing 10 000 to 30 000 events were recorded and analyzed with CellQuest software (Becton Dickinson). Intracellular cytokine staining was performed as following. Briefly, 2 × 106 freshly isolated tumor-derived cells in 1 ml RPMI 10% FCS were incubated with brefeldin A (1 µg/ml) at 37°C for 6 hr. Cells were washed; stained with surface Abs; fixed, permeabilized; and incubated with the CXCL9 or CCL5 Ab. Cells were fixed with 1% paraformaldehyde solution in PBS and analyzed on a FACS-Canto flow cytometer, using Diva software (Becton Dickinson). IFN-γ ELISA was performed using NIB42 as capture Ab and biotinylated 4S.B3 as detection Ab (source) in the concentrations described by the manufacturer. For detection of CXCL9, ELISA was done using cell supernatants as per the instructions from the supplier.
Cellquest 3 2 1 f1
Manufactured by BD
The CELLQuest 3.2.1.f1 software is a data acquisition and analysis tool for flow cytometry. It provides the core functionality to control flow cytometer instruments and analyze the resulting data.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur cytometer, by BD (3 mentions)
Alexa fluor 488 goat anti human antibody, by Thermo Fisher Scientific (2 mentions)
Facscanto, by BD (1 mentions)
Diva software, by BD (1 mentions)
Cellquest software, by BD (1 mentions)
Facscanto 2 cytometer, by BD (1 mentions)
Goat anti human alexa 488 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Lsm 510 meta confocal microscope, by Zeiss (1 mentions)
Draq5, by Cell Signaling Technology (1 mentions)
Rhodamine labelled phalloidin, by Merck Group (1 mentions)
4 protocols using cellquest 3 2 1 f1
1
Comprehensive Immune Profiling of Tumor Cells
2
EGFR, ErbB-2, and ErbB-3 Expression Analysis
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EGFR, ErbB-2 and ErbB-3 cell surface expression was analyzed by flow cytometry using a FACSCalibur cytometer (Becton Dickinson, Buccinasco, MI, Italy). Briefly, around one million of proliferating cells were harvested and labeled with cetuximab for EGFR, trastuzumab for ErbB-2 or EV20 for ErbB-3 and left on ice for 20 min. Cells were then washed with 2 ml PBS, pulled down and stained with an Alexa Fluor 488 Goat anti-Human antibody (Molecular Probes, Life Technologies, Paisley, UK). The data were analyzed using CELLQuest 3.2.1.f1 software (Becton Dickinson).
3
Endosialin and Cell Cycle Analysis
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Endosialin cell surface expression was analyzed by flow cytometry using a FACSCalibur cytometer (Becton Dickinson, Buccinasco, MI, Italy). Briefly, around one million of proliferating cells were harvested and labeled with hMP-E-8.3 on ice for 20 minutes. Cells were then washed with 2 ml PBS, pulled down, and stained with an Alexa Fluor 488 Goat anti-Human antibody (Molecular Probes, Life Technologies, Paisley, UK). The data were analyzed using CELLQuest 3.2.1.f1 software (Becton Dickinson).
Cell Cycle was analyzed by flow cytometric evaluation of DNA content according to the Nicoletti method [27 (link)]. 2×106 cells were harvested by centrifugation at 800g for 5 min and fixed in 70% ethanol at 4°C overnight. Cells were then washed twice with PBS and stained in 0.5 ml PBS containing Propidium iodide (PI, 50 μg/ml) and RNAase (10 μg/ml) 4°C overnight in the dark. 20,000 singlet gated events were acquired using a FACSCanto II cytometer (Becton Dickinson, Buccinasco, MI, Italy). Finally, data were analyzed using FLOW JO, LLC software.
Cell Cycle was analyzed by flow cytometric evaluation of DNA content according to the Nicoletti method [27 (link)]. 2×106 cells were harvested by centrifugation at 800g for 5 min and fixed in 70% ethanol at 4°C overnight. Cells were then washed twice with PBS and stained in 0.5 ml PBS containing Propidium iodide (PI, 50 μg/ml) and RNAase (10 μg/ml) 4°C overnight in the dark. 20,000 singlet gated events were acquired using a FACSCanto II cytometer (Becton Dickinson, Buccinasco, MI, Italy). Finally, data were analyzed using FLOW JO, LLC software.
4
Microscopic Evaluation of hMP-E-8.3 Binding
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SJSA-1 cells were plated in 12 well-plates and grown in 10% FBS RPMI-1640 for 24 hours. Cells were then incubated with 10 μg/ml of hMP-E-8.3 for 30 minutes on ice and returned at 37°C for 2 hours. After 2 hours, cells were stained with a goat anti-human Alexa 488-conjugated secondary antibody (ThermoFisher Scientific; Waltham, Massachussets, USA) and samples were analyzed by flow cytometry using a FACSCalibur cytometer (Becton Dickinson, Buccinasco, MI, Italy). Finally, data were analyzed using CELLQuest 3.2.1.f1 software (Becton Dickinson). For confocal imaging, after 0.5 or 2 hours, cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.25% Triton-X100 in PBS for 5 minutes, and blocked with 5% goat serum in PBS for 30 minutes at room temperature. Then cells were stained for 1 hour at room temperature with a goat anti-human Alexa 488-conjugated secondary antibody (Molecular Probes, Life Technologies, Paisley, UK) (green staining) and with Rhodamine-labelled phalloidin (Sigma-Aldrich Corporation, St. Louis, MO, USA). Cell nuclei were counterstained in blue using DRAQ5 (Cell Signaling Technology, Danvers, MA, USA). Images were acquired with a Zeiss LSM 510 meta-confocal microscope (Zeiss, Oberkochen, Germany) using 488-, 543-, and 633-nm lasers.
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