Adherent cells (OV-90, SK-OV-3, Caov-3, NIH-3T3, and HEK293) were washed twice with DPBS and harvested using Cell Dissociation Buffer (Gibco) by incubating at 37 °C, for 5 min. The suspended cells (Daudi) were harvested and washed twice with DPBS. The cells (2 × 105 cells per sample) were blocked with blocking buffer (cold DPBS containing 5% bovine serum albumin (BSA)) on ice for 1 h. The cells were then centrifuged at 1000× g and 4 °C for 3 min and stained with primary antibodies against c12G1, hIgG isotype (Thermo Fisher Scientific), c12G1-DM1, and hIgG-DM1, all diluted in wash buffer (cold DPBS containing 2% BSA). The cells were then washed twice with wash buffer and stained with the secondary antibody on ice for 1 h. Secondary antibodies, including Alexa Fluor™ 488-conjugated anti-mouse IgG (0.5 μg/mL; Thermo Fisher Scientific) and fluorescein isothiocyanate-conjugated anti-human IgG (0.3 μg/mL; Thermo Fisher Scientific), were diluted in wash buffer. Fluorescence signals were detected using CyFlow Cube 6 (Sysmex Partec, Gorlitz, Germany) and analyzed using the FCS Express™ 6 flow cytometry software (De Novo Software, Los Angeles, CA, USA).
Fcs express 6 flow cytometry software
Manufactured by De Novo Software
Sourced in United States
FCS Express 6 Flow Cytometry Software is a data analysis platform designed for flow cytometry research. It provides tools for visualizing and analyzing data obtained from flow cytometry experiments.
Automatically generated - may contain errors
Lab products found in correlation
Flow cytometry staining buffer, by Thermo Fisher Scientific (2 mentions)
Ze5 cell analyzer, by Bio-Rad (2 mentions)
Caspase 3 7 assay kit, by Thermo Fisher Scientific (2 mentions)
Fcs express multicycle, by De Novo Software (2 mentions)
Annexin 5 fitc, by Dojindo Laboratories (2 mentions)
Cell dissociation buffer, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Cyflow cube 6, by Sysmex (1 mentions)
Propidium iodide, by Cayman Chemical (1 mentions)
Lsr 2 flow cytometry analyzer, by BD (1 mentions)
4 oht, by Merck Group (1 mentions)
Cell strainer, by BD (1 mentions)
70 μm cell strainer, by Avantor (1 mentions)
Anti cd19 pe cy7, by BD (1 mentions)
Lsr flow cytometer, by BD (1 mentions)
Anti cd3e v450 500a2, by BD (1 mentions)
Rbc lysis buffer, by BioLegend (1 mentions)
Excel 2018, by GraphPad (1 mentions)
Nis elements imaging software, by Nikon (1 mentions)
Polybrene, by Thermo Fisher Scientific (1 mentions)
15 protocols using fcs express 6 flow cytometry software
1
Flow Cytometry Analysis of Antibody-Drug Conjugates
2
Flow Cytometry Analysis of Cell Treatment
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Flow cytometry analysis was performed as previously described [11 (link),16 (link),39 (link)]. Briefly, MCF-7 and BT474 cells were seeded at a density of 1 × 106 cells/plate and their media was changed two times in every two days. Following an overnight treatment with 4-OHT (10−6 M) (Sigma) and SEL (10−7 M) (Selleckchem) alone and in combination, cells were collected with 0.1% FBS containing 1× PBS, washed, resuspended at 1–2 × 106 cells/mL and fixed with ice cold ethanol for 24 h. Next day, cells were washed with 1× PBS and incubated in 10 ng
RNAase for one hour at room temperature. Cells were stained with 0.25% Propidium Iodide (#10008351, Cayman Chemicals, Ann Arbor, MI, USA) for one hour and results were analyzed by BD™ LSR II Flow cytometry analyzer (BD Biosciences Inc., San Jose, CA, USA). Following analysis, results were analyzed by FCS Express 6 Flow Cytometry Software (DeNovo Software,https://www.denovosoftware.com/site/Flow-RUO-Overview.shtml ) and all experiments were repeated three times.
RNAase for one hour at room temperature. Cells were stained with 0.25% Propidium Iodide (#10008351, Cayman Chemicals, Ann Arbor, MI, USA) for one hour and results were analyzed by BD™ LSR II Flow cytometry analyzer (BD Biosciences Inc., San Jose, CA, USA). Following analysis, results were analyzed by FCS Express 6 Flow Cytometry Software (DeNovo Software,
3
Quantification of Cellular Uptake of PNAs
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HeLa pLuc/705 cells were seeded in 24 well plates at 1.0 × 105 cells/well the day before PNA treatment. PNA solutions were prepared at 10× concentration as described in the PNAs antisense activity and toxicity section. 50 μL of PNA solution was added to wells containing 500 μL full medium and mixed well. Cells were incubated for 4 h, and then washed three times with pre-warmed full medium. Cells were detached from plates by gently pipetting with phosphate buffered saline (PBS) containing 1% bovine serum albumin (BSA). Cells were then filtered with a cell strainer (70 μm, BD Falcon) and subjected to flow cytometric analysis (30,000 events for each sample) using Accuri C6 Personal Flow Cytometry (BD Biosciences, Lyngby, Denmark). The mean fluorescence signal was calculated by FCS Express 6 Flow Cytometry Software (De Novo Software, Glendale, CA, USA).
4
Apoptosis and Cell Cycle Analysis by Flow Cytometry
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Cells for flow cytometry were counted and 5×105-1×106 cells were placed in individual 12×75 mm round-bottom tubes suspended in flow cytometry staining buffer (Invitrogen, 00-4222-26). For apoptosis, a Caspase-3/7 assay kit was used (Invitrogen, C10427). Each tube was brought to a volume of 1 ml staining buffer and 1 μl of CellEvent Caspase-3/7 reagent was added. Cells were incubated at 37 °C in the dark for 25 minutes and then 1μl of SYTOX AADvanced dead stain solution was added. Cells were incubated at 37 °C for 5 minutes and then analyzed. For cell cycle analysis, cells were fixed with 0.5 ml of 100% cold ethanol for 20 minutes at 4 °C under gentle rotation. Cells were pelleted and the ethanol was decanted. Cells were resuspended in 1 ml of staining buffer and 4 drops (164 μl) of Propidium lodide Ready Flow (Invitrogen, R37169) was added to the cells and incubated at 25 °C for 20 minutes. Cells were analyzed on a ZE5 Cell Analyzer (Bio-Rad) and data processed using FCS Express 6 Flow Cytometry software (De Novo Software, Pasadena, CA). The percentage of the cell population in G1, or S/G2 phase of the cell cycle was calculated by fitting Propidium Iodide excitation counts using FCS Express: Multicycle (De Novo Software).
5
Immune Cell Isolation from Murine MLN
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Mechanical digestion of the MLN was achieved by a syringe plunger. After mechanical digestion, single cells were obtained by passing the cells through a 70-μm cell strainer (VWR International, Radnor, PA). Red blood cells (RBC) were lysed using RBC lysis buffer (BioLegend, San Diego, CA). Cells from 2 mice in the same experimental groups were combined to increase cell numbers for analyses. For T-cell populations, cells (1 × 106 cells) were stained with antibodies anti–CD3e-V450 (500A2; BD Biosciences, Franklin Lakes, NJ) and anti–CD4-PE-Cy5 (RM4-5; BD Biosciences). For B-cell populations, cells (1 × 106 cells) were labeled with anti–CD19-PE-Cy7 (1D3; BD Biosciences) and anti–MHC-II-FITC (2G9; BD Biosciences). Populations of T and B cells then were determined by a BD LSR flow cytometer (BD Biosciences ). Flow cytometry data were analyzed using FCS Express 6 Flow Cytometry Software (De Novo Software, Glendale, CA). The gating strategy used to determine immune cell populations is presented in Supplemental Figure 1 .
6
Quantitative Cell Imaging and Analysis
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The number of the used animals and independent experiments are labeled in the Figure legends. All macroscopic pictures and microscopic images are representative of the indicated number of independent experiments. Individual data points with mean and SEM or median and interquartile range (IQR) are shown in each diagram. Box plots are shown with whiskers of 10–90 percentiles. Microscopic image processing and analysis were performed using NIS-Elements Imaging Software (Nikon, version BR 4.60.00), Photoshop CS6 (Adobe, version 16.0.0), and NIH Fiji software (versions 1.52i -1.53 f). Flow cytometry result processing and analysis were performed using FCS Express 6 Flow Cytometry Software (De Novo Software, version 6.06.0033. Data processing and statistical analyses were performed using Graphpad Prism (version 7.03) and Excel 2018 software. All datasets were tested for normality using Shapiro–Wilk test of normality. A dataset was considered to be normally distributed with P > 0.01. Normally distributed datasets were tested with Student paired or unpaired two-tailed T-test and non-normally distributed datasets were tested with Wilcoxon signed-rank test. A difference was considered statistically significant at P < 0.05.
7
Determination of Lentiviral Titers by Flow Cytometry
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Virus-containing supernatant was harvested from iSLK.219 cells at the indicated times by pelleting cellular debris at 3300 x g for 5 minutes and then stored at -80°C until ready to titer the virus. 105 293A cells/well were seeded in 12-well plates to obtain a confluent monolayer two days later. The thawed viral inoculum was briefly vortexed and centrifuged again at 3300 x g for 5 minutes. Two-fold serial dilutions of viral supernatants were applied to the monolayer containing 4 μg/mL polybrene and 25 mM HEPES (Gibco) and centrifuged at 800 x g for 2 h at 30°C. The total cell count per well was also determined from an uninfected well. Fresh media was applied immediately after spinoculation. 20–24 h post-infection, two dilutions that resulted in less than 30% GFP-positive cells (the linear range for infection) were trypsinized, washed once with PBS and fixed with 1% paraformaldehyde in PBS. GFP-positivity was measured on either FACSCalibur or FACSCanto cytometers (BD) by gating on FSC/SSC and counting 10000–15000 “live” events. Gating and % GFP positive events were determined with FCS Express 6 Flow Cytometry Software (ver.6.0; De Novo). Virus titer was calculated as IU/mL with the following formula:
The virus titer of the two dilutions were averaged for the final titer value.
The virus titer of the two dilutions were averaged for the final titer value.
8
HUVEC Liposome Binding Assay
9
Analyzing Apoptosis in lncRNA CASC2 Overexpression
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The overexpression rate of lncRNA CASC2 reached 200% at 24 h after transfection. Therefore, cell apoptosis was detected by cell apoptosis assay at this time point. Serum-free cell culture medium was used to prepare single-cell suspensions with a cell density of 5×104 cells/ml. The cell suspension was transferred to a 6-well plate with 2 ml in each well. Cells were cultivated at 37°C for 48 h and were then digested with 0.25% trypsin. After that, Annexin V-FITC (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) and propidium iodide staining was performed at 4°C for 30 min, and apoptotic cells were detected by flow cytometry. Data were analyzed using FCS Express 6 Flow Cytometry Software (De Novo Software, Glendale, CA, USA).
10
Flow Cytometry Analysis of Apoptosis and Cell Cycle
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Cells for flow cytometry were counted and 5×105–1×106 cells were placed in individual 12 × 75 mm round-bottom tubes suspended in flow cytometry staining buffer (Invitrogen, 00–4222-26). For apoptosis, a Caspase-3/7 assay kit was used (Invitrogen, C10427). Each tube was brought to a volume of 1 ml staining buffer and 1 μl of CellEvent Caspase-3/7 reagent was added. Cells were incubated at 37 °C in the dark for 25 minutes and then 1 μl of SYTOX AADvanced dead stain solution was added. Cells were incubated at 37 °C for 5 minutes and then analyzed. For cell cycle analysis, cells were fixed with 0.5 ml of 100% cold ethanol for 20 minutes at 4 °C under gentle rotation. Cells were pelleted and the ethanol was decanted. Cells were resuspended in 1 ml of staining buffer and 4 drops (164 μl) of Propidium Iodide Ready Flow (Invitrogen, R37169) was added to the cells and incubated at 25 °C for 20 minutes. Cells were analyzed on a ZE5 Cell Analyzer (Bio-Rad) and data processed using FCS Express 6 Flow Cytometry software (De Novo Software, Pasadena, CA). The percentage of the cell population in G1, or S/G2 phase of the cell cycle was calculated by fitting Propidium Iodide excitation counts using FCS Express: Multicycle (De Novo Software).
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