Multiparameter flow cytometry (MFC) and sample processing was carried out as described previously [70 (link)]. MFC analyses were performed using FC500 or Navios flow cytometers (Beckman Coulter, Miami, FL, USA). List mode files were analyzed using CXP Software version 2.0 and Kaluza version 1.0 (Beckman Coulter, Brea, CA, USA). Diagnoses were assigned according to EGIL and WHO classifications [10 ,71 (link)]. Single cell suspensions of transduced c-Kit+ cells were prepared as described elsewehere [15 (link)]. Dead cells were excluded by gating on 7AAD (Miltenyi Biotec)-negative cells. Flow cytometry analysis were performed on an LSR Fortessa cell analyser (BD Biosciences, San Jose, CA, USA) and data were analysed with FlowJo software v 10 (BD, Franklin Lakes, NJ, USA).
Cxp software version 2
Manufactured by Beckman Coulter
Sourced in United States
CXP Software version 2.2 is a data analysis software suite designed for Beckman Coulter flow cytometry instruments. It enables users to acquire, analyze, and report data from their flow cytometry experiments.
Automatically generated - may contain errors
Lab products found in correlation
Cytomics fc500 flow cytometer, by Beckman Coulter (6 mentions)
Cytomics fc500, by Beckman Coulter (3 mentions)
Fc500, by Beckman Coulter (2 mentions)
Lsrfortessa cell analyser, by BD (1 mentions)
Navios, by Beckman Coulter (1 mentions)
7 aad, by Miltenyi Biotec (1 mentions)
2 7 dichlorofluorescein diacetate dcfh da, by Merck Group (1 mentions)
Propidium iodide, by BD (1 mentions)
Fc500 flow cytometry, by Beckman Coulter (1 mentions)
Fitc annexin 5 apoptosis detection kit, by BD (1 mentions)
Rotor gene q real time pcr machine, by Qiagen (1 mentions)
Rnase a, by Merck Group (1 mentions)
Tryple express, by Thermo Fisher Scientific (1 mentions)
Dcf da, by Thermo Fisher Scientific (1 mentions)
Cd45 pe cy7, by BioLegend (1 mentions)
Cd33 pe cy5, by BioLegend (1 mentions)
Cxcr4, by BD (1 mentions)
Cx3cr1, by BioLegend (1 mentions)
Anti foxp3 mab, by Thermo Fisher Scientific (1 mentions)
Anti cd19 mab, by Thermo Fisher Scientific (1 mentions)
14 protocols using cxp software version 2
1
Multiparameter Flow Cytometry for Cell Analysis
2
Immunophenotyping of Adipose-Derived Stem Cells
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The expression of cell-associated surface markers was measured by flow cytometric analysis. In brief, ADSCs of P5 were collected by standard trypsin digestion, and the cells were fixed and permeabilized in 70% (v/v) ice-cold ethanol for 12 h. The samples were blocked with 10% (v/v) goat serum for 30 min at room temperature and were incubated in 1% BSA containing rabbit anti-chicken polyclonal antibodies to CD29, CD44, CD71 and CD73 (all at 1:100 dilution) for 1 h at room temperature. Primary antibody was replaced with PBS for the negative control. Subsequently, the samples were washed with PBS and incubated in PBS containing FITC-conjugated goat anti-rabbit IgG as the secondary antibody for 1 h at room temperature. After washing with PBS, the labeled cells were detected with a flow cytometer. Flow cytometric data were analyzed using CXP software version 2.0 (Beckman Coulter, Brea, CA, USA).
3
BM and LPS Effects on RAW264.7 Cells
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RAW264.7 cells (1×106/well) were synchronized in G1 phase by serum deprivation, and different concentrations of BM (25, 50 and 100 μg/ml) and 1 μg/ml LPS were then added. Cells were harvested after 48 h. Propidium iodide (PI)-stained single-cell suspension was analyzed on a FC5000 cytometer (BD Biosciences, Franklin Lakes, NJ, USA) using CXP software version 2.0 (Beckman Coulter, Fullerton, CA, USA) for data analysis. The experiment was repeated three times. For apoptosis analysis, following BM treatment (25, 50 and 100 μg/ml) for 48 h, cells were stained with Annexin V and PI (Clontech Laboratories, Inc., Mountainview, CA, USA) for 15 min at room temperature. The apoptotic index was determined by flow cytometry with Flowjo software. 2′7′-Dichlorofluorescein diacetate (DCFH-DA; Sigma-Aldrich; Merck KGaA) was used to measure ROS formation. After exposure to different concentrations of BM (25, 50 and 100 μg/ml) for 48 h, the cells were then incubated in DCFH-DA-containing medium (final concentration, 10 μM) at 37°C for 20 min. The cells were washed with PBS three times to remove DCFH-DA that had not entered into the cells. The intracellular concentration of DCFH-DA was then measured by flow cytometry.
4
Cell Cycle Analysis by Flow Cytometry
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Following transfection with plasmids or treatment with TSN as aforementioned, cells were washed twice with PBS and detached from the plate surface by digestion using trypsin. Cells were centrifuged at 300 × g at 4°C for 10 min, the pellet was resuspended in PBS, centrifuged again at 300 × g at 4°C for 10 min and resuspended in ice-cold 70% ethanol and stored at 4°C for 18 h. Samples were washed once in PBS and resuspended in DNA staining solution (propidium iodide, 5 µg/ml; RNase A, 0.5 mg/ml; PBS) and incubated at 37°C in the dark for 30 min. All samples were assessed using a Cytomics FC500 Flow Cytometer (Beckman Coulter, Inc.) and analyzed using CXP Software version 2.3 (Beckman Coulter, Inc.).
5
Apoptosis Analysis of Metformin and Everolimus
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CaSki and C33A cells were seeded into 6-well plates at a density of 1 × 106 cells per well and exposed to 10 mM metformin with or without 20 μM everolimus for 48 h. Cells were then collected and fixed with 70% ice-cold ethanol at −20 °C overnight. After fixation, cells were centrifuged at 400× g for 10 min at 4 °C, washed with cold PBS, and stained with 0.5 mL PI/RNases staining buffer (PI, 10 μg/mL; RNases, 300 μg/mL; #550825, BD Pharmingen; BD Biosciences, Franklin Lakes, NJ, USA) for 15 min at room temperature in the dark. Cell apoptosis was detected using an FITC Annexin V apoptosis detection kit (#556547, BD Biosciences); cells were double-stained with 5 μL FITC Annexin V (20 μg/mL) and 5 μL propidium iodide (PI, 50 μg/mL) for 15 min at RT in the dark. Finally, the stained cells were analyzed using FC500 flow cytometry and CXP software (version 2.3; Beckman Coulter, Brea, CA, USA), and early and late apoptotic or necrotic cells were assessed.
6
Flow Cytometry and Viral Load Analysis
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CD4+ and CD8+ cells were quantified by flow cytometry (Cytomics FC500; Beckman Coulter, Inc.) with CXP Software (version 2.3; Beckman Coulter, Inc.). The viral load was determined in plasma using Arthus® HI Virus-1 QS-RGQ kit (Qiagen Inc.), QIAsymphony® SP/AS sample extraction (Qiagen Inc.) and preparation apparatus and Rotor-Gene Q® real-time PCR machine (Qiagen Inc.). Conditions: reverse transcription of the RNA was done for 30 min at 50°C; initial activation of the hot-start enzyme was for 15 min at 95°C; amplification of the cDNA (denaturation: 95°C for 30s, annealing: 50°C for 60s and elongation; 62°C for 30s) for 50 cycles. Genotyping and miRNA analysis were performed for 1–2 days and 12–36 months following plasma extraction, respectively.
7
Cell Cycle Analysis by Flow Cytometry
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Collected cells were washed twice with cold PBS, fixed with 70% ethanol for 1 h at 4 °C, treated with 1 mg/mL of RNase A (Sigma-Aldrich, St. Louis, MO, USA), and then stained with 50 μg/mL of PI (Sigma-Aldrich). Data were acquired using a Cytomics FC500 Flow Cytometer equipped with two laser sources (Beckman Coulter, Brea, CA, USA). The results were analyzed using CXP Software version 2.2 (Beckman Coulter).
8
ROS Quantification by DCF-DA Cytometry
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Intracellular generation of ROS was measured using 2′,7′-dichlorodihydrofluorescene diacetate (DCF-DA; Molecular Probes, Eugene, OR, USA). Cells were stained with 5 μM of DCF-DA in a serum-free medium for 15 min and removed from the plate using trypLE-Express (Gibco, Waltham, MA, USA). Fluorescence intensity was measured using a Cytomics FC500 flow cytometer (Beckman Coulter) with an excitation wavelength of 480 nm and an emission wavelength of 525 nm. Data were analyzed using CXP Software version 2.2 (Beckman Coulter).
9
Quantification of HSP90 Expression in PBMCs
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Cells were stained for surface antigens CD33 PE/Cy5 and CD45 PE/Cy7 (clones WM33 and HI30, respectively, both from BioLegend, San Diego, CA, USA), fixed with paraformaldehyde, permeabilized with saponin (Beckman Coulter, Immunotech, Marseille, France), and incubated with phycoerythrin (PE)—conjugated anti-HSP90a monoclonal antibodies (Enzo Life Sciences, Ann Arbor, MI, USA) at a 1:10 (v/v) dilution. Flow cytometric analysis was performed (a) within 1 h of blood sampling (subjunctive’s baseline) and (b) 4 h after induction on an FC-500 instrument (Beckman Coulter, Miami, FL, USA) using CXP software version 2.2 (Beckman Coulter, Miami, FL, USA). Identification of PBMCs was achieved by the expression of CD45 compared to sideward scattering. Monocyte (CD33 ++ CD45 + SS med) and lymphocyte (CD33 − CD45 ++ SS low) populations were further identified based on CD33 and CD45 expression intensity. HSP90 intracellular expression was assessed in each cell type using mean fluorescence intensity (MFI). We performed instrumental quality control routinely to adjust acquisition settings to any photomultiplier (PMT) voltage alterations.
10
Phenotype Analysis of Expanded Cells
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The phenotype of expanded cells and PBMCs at baseline (day 0), before the 3rd administration (day 14), and 4 weeks after 3rd administration (day 42) were analyzed by flow cytometry. Monoclonal antibodies specific for CD3, CD16, CD56 (Beckman Coulter, CA, USA), NKG2D (eBioscience, CA, USA), CXCR3 (R&D system, MN, USA), CXCR4 (Becton–Dickinson, CA, USA), CX3CR1 (Bio Legend, CA, USA) were applied. Each monoclonal antibody was conjugated as follows: CD3 CD16, CD69, CXCR3 with fluorescein isothiocyanate (FITC)-, CD56, NKG2D, CXCR4, CX3CR1 with phycoerythrin (PE)-, CD3 with phycoerythrin-Cyanin 5 (PE-Cy5)-, CD56 with phycoerythrin-Cyanin 7 (PC7-Cy7)-. Cells were analyzed by Cytomics FC500 (Beckman Coulter) and data were acquired by the CXP software, version 2.2 (Beckman Coulter) according to the manufacturer’s instruction.
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