SK-HEP1 cells were treated at indicated concentrations of Bu or Treo for 8 hours. Reactive oxygen species (ROS) levels were determined by incubating the cells with DCFDA for 15 min, followed by acquiring the cells in a Gallios flow cytometer (Beckman Coulter, USA). The data were analyzed using Kaluza software version 2.0 (Beckman Coulter, USA). Inhibition of ROS was analyzed by pre-treating the cells with N-acetyl cysteine (NAC) (1mM) or metformin (2mM or 5mM) for 1 hour prior to Bu treatment.
Kaluza software version 2
Manufactured by Beckman Coulter
Sourced in United States
Kaluza software version 2.1 is a data analysis software developed by Beckman Coulter. It is designed to enable efficient processing and visualization of flow cytometry data.
Automatically generated - may contain errors
Lab products found in correlation
Gallios flow cytometer, by Beckman Coulter (2 mentions)
Cytoflex flow cytometer, by Beckman Coulter (2 mentions)
Cytoflex, by Beckman Coulter (2 mentions)
Navios cytometer, by Beckman Coulter (2 mentions)
Facsverse, by BD (2 mentions)
Navios flow cytometer, by Beckman Coulter (2 mentions)
Pharm lyse lysing buffer, by BD (1 mentions)
Fluorescence latex beads, by Merck Group (1 mentions)
Cytomics fc500 flow cytometer, by Beckman Coulter (1 mentions)
Annexin 5 fitc early apoptosis detection kit, by Cell Signaling Technology (1 mentions)
Cycle phase determination kit, by Cayman Chemical (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Z vad fmk, by R&D Systems (1 mentions)
Fitc annv, by Immunotools (1 mentions)
Jmp 14, by SAS Institute (1 mentions)
Click it plus edu alexa fluor 647 flow cytometry kit, by Thermo Fisher Scientific (1 mentions)
P3566, by Thermo Fisher Scientific (1 mentions)
Rnase a, by Roche (1 mentions)
Annexin 5 kit, by Miltenyi Biotec (1 mentions)
Click it plus edu alexa fluor 647 flow cytometry assay kit, by Thermo Fisher Scientific (1 mentions)
30 protocols using kaluza software version 2
1
Evaluating Cellular ROS Levels Under Bu and Treo
2
Cell Cycle Analysis of SK-HEP1 Cells
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SK-HEP1 cells were treated with either Bu (300 μM) alone or pre-treated with metformin (2mM) or U0126 (10 μM) before Bu treatment. The cells were fixed and permeabilized using methanol, followed by incubation with DAPI (10 μg/ml). Cell cycle status was determined by acquiring the cells in a Gallios flow cytometer (Beckman Coulter, USA), and data were analyzed using Kaluza software version 2.0 (Beckman Coulter, USA).
3
Identification of Monocyte Subsets
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Monocyte subpopulations were identified with flow cytometry using the lysis-no-wash strategy (BD Pharm Lyse lysing buffer, Becton Dickinson) on fresh EDTA blood. A total of 100 μl of EDTA blood was stained by monoclonal antibodies (CD16 FITC NKP15 Becton Dickinson, CD14 PE RMO52 Beckman Coulter, HLA-DR Immu357 PC5.5 Beckman Coulter, and CD45 PC7 J33 Beckman Coulter). Surface expression was assessed using FC500 and CytoFLEX flow cytometer (Beckman Coulter) and analyzed with Kaluza software version 2.1 (Beckman Coulter). The applied gating strategy was in short; monocytes were selected in the SSC/CD45+ plot, gated to SSC/HLA-DR + plot, identifying monocytes as CD45+ HLA-DR + cells with monocyte scatter properties. Exclusion of lymphocytes, and natural killer cells was performed by excluding CD45+ HLA-DR+ CD14– CD16– cells. In the CD14/CD16 plot, the percentages of gated monocyte subsets (classical CD14++CD16−), intermediate (CD14++CD16+), and nonclassical monocytes (CD14+CD16++) were used for analyses. Identification of monocytes subsets follows current recommendations (21 (link)).
4
Multipotency and RPE Differentiation of miPB-IPCs
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To determine miPB-IPC’s multipotency and their RPE cell differentiation (Figure S1 ), miPB-IPC were treated with combined supplements (including L-glutamine, Gentamicin sulfate-Amphotericin (GA-1000), and basic fibroblast growth factor) in the presence of retinal pigment epithelial growth media (Lonza) for 8 days, in 24-well tissue culture-treated plates, at 37 °C in 5% CO2. The differentiated cells were characterized by immunocytochemistry with RPE-specific markers such as mouse anti-human mAbs RPE 65, CRALBP, and claudin-19, along with rabbit anti-tight junction protein 1 (ZO-1) polyclonal Ab (Novus Biological, Littleton, CO, USA). Human primary RPE cells were purchased from Lonza and served as positive control. Isotype-matched IgG served as negative control for immunostaining. For functional analysis, the phagocytosis of fluorescence latex beads (Sigma, Saint Louis, MO, USA) were performed in differentiated RPE cells. The phagocytosis-associated surface marker CD36 was examined by flow cytometry. The level of CD36 expression was quantified by mean fluorescence intensity after analyzed with Kaluza software version 2.1 (Beckman Coulter).
5
Kinetics of Peptide Binding to Bacteria
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The penetration activity of P7 with bacterial cell membranes at different time interval was investigated by flow cytometry (Zhang et al., 2016 (link)). The mid-log bacterial suspensions (approximately 107 CFU/ml in 1 × PBS) were incubated with 1 × MIC of TAMRA-labeled P7 at 37°C for 5, 10, 15, 30, 60, or 120 min. Subsequently, unbound labeled peptide was discarded by washing with 1 × PBS. The red fluorescence (585/42 nm) signal emitted by TAMRA fluorescence dye was evaluated by flow cytometry (CytoFlex, Beckman Coulter). 25,000 bacterial cells were counted in each sample. The experiments were done in triplicate and the data was analyzed by Kaluza software version 2.1 (Beckman Coulter, Brea, CA, United States).
6
Evaluating Gal-9 Induced Apoptosis and Cell Cycle
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We conducted a flow cytometric analysis using the Cycle Phase Determination kit (Cayman Chemical Co.) to evaluate the mechanism of growth inhibition by Gal-9. CW-2 cells were digested with 0.25% trypsin and plated in 100-mm-diameter dishes at 1.0×106 cells per dish. After incubation for 24 h without FBS, CW-2 cells were treated with 0.3 µM of Gal-9 or dimethyl sulfoxide (DMSO; control) for another 24 h, then harvested, washed with phosphate-buffered saline (PBS), suspended in 500 µl of PBS plus 10 µl of RNase A (250 µg/ml) and 10 µl of propidium iodide (PI) stain (100 µg/ml), and incubated for 30 min.
To determine the apoptosis rate of CW-2, CACO-2, and WiDr cells, we used flow cytometry and the Annexin V-FITC Early Apoptosis Detection Kit (Cell Signaling Technology, Inc.). CW-2, CACO-2 and WiDr cells were plated in 100-mm-diameter dishes at 1.0×106 cells per dish and treated with 0.3 µM Gal-9 or DMSO control for 24 h. After incubation for 24 h, CW-2, CACO-2 and WiDr cells were harvested, and washed with PBS. Staining was performed according to the manufacturer's protocol. After adding Annexin V-FITC and PI, we analyzed apoptosis and necrotic cell death. Flow cytometry was conducted with a Cytomics FC 500 flow cytometer (Beckman Coulter) equipped with a 480-nm argon laser. Cell percentages were analyzed with Kaluza software version 2.1 (Beckmann Coulter).
To determine the apoptosis rate of CW-2, CACO-2, and WiDr cells, we used flow cytometry and the Annexin V-FITC Early Apoptosis Detection Kit (Cell Signaling Technology, Inc.). CW-2, CACO-2 and WiDr cells were plated in 100-mm-diameter dishes at 1.0×106 cells per dish and treated with 0.3 µM Gal-9 or DMSO control for 24 h. After incubation for 24 h, CW-2, CACO-2 and WiDr cells were harvested, and washed with PBS. Staining was performed according to the manufacturer's protocol. After adding Annexin V-FITC and PI, we analyzed apoptosis and necrotic cell death. Flow cytometry was conducted with a Cytomics FC 500 flow cytometer (Beckman Coulter) equipped with a 480-nm argon laser. Cell percentages were analyzed with Kaluza software version 2.1 (Beckmann Coulter).
7
Treg Cell Monitoring by Flow Cytometry
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Flow cytometry was performed on day 0, day 15, and on day of infusion to evaluate the cell population and identify Treg (Figure S1 ). The multicolor panel was composed of CD3 (FITC), CD4 (PE), CD8 (APC), CD25 (PE‐Cy7), CD127 (APC‐R700), CD19 (BV510), and 7‐AAD cell viability solution. Data were acquired with a Gallios Flow Cytometer (Beckman Coulter, Brea, California) and analyzed with Kaluza software version 2.1 (Beckman Coulter). Cell debris and doublets were excluded using forward scatter (FSC) and side scatter (FSC) parameters, and dead cells were excluded on the basis of 7‐aminoactinomycin D (7‐AAD) staining. Treg cells were identified as CD3+CD4+CD25+CD127dim cells.
8
Sperm Viability Assessment by Flow Cytometry
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Sperm viability and data analysis were performed using the flow cytometry system described above and Kaluza® software, Version 2.1 (Beckman Coulter Ltd.). Frozen-thawed semen samples were diluted in SP-Talp media (105 mM NaCl, 3.1 mM KCl, 0.4 mM MgCl2, 2.0 mM CaCl2⋅2H2O, 0.3 mM NaH2PO4⋅H2O, 1 mM sodium pyruvate, 21.6 mM sodium lactate, 20 mM Hepes, 20 mM Hepes salt, 5 mM glucose, 50 μg/ml gentamycin) to a concentration of 1 × 106 sperm cells/ml. Two technical replicates were considered per sample. Sperm suspensions were stained with 0.48 μM propidium iodide (PI, Sigma-Aldrich) and incubated for 10 min prior to flow cytometric analysis. PI fluorescence was detected using a 670 nm long pass filter (FL3), and gating was performed to reveal sperm cells population (based on electronic volume) and percentages of living spermatozoa as previous described (Standerholen et al., 2014 (link)).
9
Apoptosis and Cell Cycle Analysis
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Cells were left untreated or treated with either control siRNA (final concentration 25 nmol/L) or IMP1 siRNA (final concentration 25 nmol/L) with or without Z-VAD-FMK (final concentration: 20 μmol; R&D Systems, Minneapolis, MN, USA). Cells were collected, washed twice in Annexin V (AnnV) buffer, stained with FITC-AnnV (final dilution: 1:100; Immunotools, Friesoyte, Germany) according to the manufacturer’s guidelines, and incubated with propidium iodide (PI) (5 mg/mL) for 30 min at 4 °C. Cell death was quantified using flow cytometry. For cell-cycle distribution, cells were untreated or transfected with CTR siRNA or IMP1 siRNA (final concentration 25 nmol/L) for 48 h and analyzed as previously indicated [11 (link)]. Cells were analyzed using flow cytometry Gallios and Kaluza software Version 2.1 (Beckman Coulter Life Sciences, Pasadena, CA, USA).
10
Multiparameter Flow Cytometry for AML MRD Detection
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For the routine diagnosis of AML as well as for controls post-chemotherapy/BM transplantation for MRD detection, the Routine Diagnostics Laboratory currently uses one to eight 10-color panels (Table S2 ). The laboratory has developed gating strategies to analyze normal and aberrant CD34pos HSPCs and define MRD positivity with a sensitivity of 0.1%, as required by the ELN [16 (link)]. For normal HSPCs, myeloid CD33pos (approx. 70% of CD34pos HSPCs) and lymphoid CD19pos (approx. 30% of CD34pos HSPC) subpopulations are defined (Figure S1A ). Subsequently, normal and aberrant lineage marker expressions are analyzed on the myeloid subpopulation (Figure S1B ) using a combination of LAIP and “different from normal” approaches [33 (link)]. The panels are run on a Navios cytometer, with the acquisition of >1 million events, and analyzed using KALUZA software Version 2.1 (Beckman Coulter, Brea, CA, USA).
To determine MRD status, the results from flow cytometry are routinely compared to results from molecular biology analysis, and a consensus result is reported. In the sixteen cases where MRD was found to be negative using flow cytometry, one case gave a positive result using NGS for RUNX1 and SF3B1 mutations and, thus, was considered to be MRD positive (Case 23;Table S1 ).
To determine MRD status, the results from flow cytometry are routinely compared to results from molecular biology analysis, and a consensus result is reported. In the sixteen cases where MRD was found to be negative using flow cytometry, one case gave a positive result using NGS for RUNX1 and SF3B1 mutations and, thus, was considered to be MRD positive (Case 23;
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