Cell cycle was analyzed with the KeyGenDNA Content Quantitation Assay (Nanjing KeyGen Biotech Co., Ltd.), following the manufacturer's protocol. A total of 3×105 cells were seeded into a well of a 6-well plate and incubated with test chemicals or 0.01% DMSO as a negative control for 24 h. Following incubation, cells were collected, washed twice with PBS and 1×106 cells/ml were fixed with 500 µl 70% cold methanol at 4°C overnight. Cells were then washed with PBS, 100 µl RNase solution was added and the plate was incubated at 37°C for 30 min. Cells were stained with 400 µl PI at 4°C in the dark for 30 min, and fluorescence intensity was analyzed by flow cytometry at 488 nm and using Novoexpress version 1.0.2 software (ACEA Biosciences Inc.). All experiments for cell cycle detection were repeated 3 times.
Novoexpress version 1
Manufactured by Agilent Technologies
Sourced in United States
NovoExpress® version 1.2.4 is a software package developed by Agilent Technologies. It is designed to provide data acquisition and analysis capabilities for flow cytometry experiments. The software supports a range of Agilent flow cytometry instruments and enables users to collect, visualize, and analyze flow cytometry data.
Automatically generated - may contain errors
Lab products found in correlation
Novocyte flow cytometer, by Agilent Technologies (8 mentions)
Keygendna content quantitation assay, by Keygen Biotech (2 mentions)
Annexin 5 fitc kit, by BD (2 mentions)
Cycletest plus dna reagent kit, by BD (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Apoptosis detection kit, by Keygen Biotech (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
100 μm cell strainer, by Corning (1 mentions)
Fix perm cell fixation and cell permeabilization kit, by Thermo Fisher Scientific (1 mentions)
Collagenase 4, by Thermo Fisher Scientific (1 mentions)
Evos fl imaging system, by Thermo Fisher Scientific (1 mentions)
2 7 dichlorofluorescein diacetate dcfh da, by Beyotime (1 mentions)
Acea flow cytometer, by Agilent Technologies (1 mentions)
Annexin 5 fitc propidium iodide staining, by BD (1 mentions)
Annexin 5 fitc pi, by Merck Group (1 mentions)
12 protocols using novoexpress version 1
1
Cell Cycle Analysis via Flow Cytometry
2
Cell Apoptosis Detection by Flow Cytometry
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A double staining assay (Apoptosis Detection kit; Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) was used, according to the manufacturer's protocol, to assess whether the detected compounds were able to induce cell apoptosis. Exponentially growing cells were seeded (3×105 cells/well) in a 6-well plate, which was incubated with different compounds for 24 h at 37°C. Cells were collected, washed twice with phosphate buffered saline (PBS), and suspended in 500 µl binding buffer. Cells were stained and incubated with 5µl Annexin V-fluorescein isothiocyanate and 2 µl propidium iodide (PI) solution homogeneously for 5–15 min in the dark at room temperature. Cell suspensions were immediately measured by flow cytometry and Novoexpress version 1.0.2 software (ACEA Biosciences Inc., San Diego, CA, USA). All experiments for apoptosis were repeated 3 times.
3
Cell Cycle Analysis of HeLa Cells
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KeyGenDNA Content Quantitation assay (Nanjing KeyGen Biotech Co., Ltd.) was performed to analyze cell cycle phase-distribution, sticking strictly to the manufacturer’s protocol. Human HeLa cervical cancer cells were seeded at a density of 3×105 cells/well in a 6-well plate. These plates were then incubated with different doses (control, 3, 6, and 12 μM) of the molecule for 12 hours. Incubation was followed with collection and washing twice with PBS. After that washed cells were fixed overnight in 70% cold methanol at 4°C. Again, washing with PBS was done. Washing was followed by addition of 100 μL RNase solution and incubating for half an hour at 25°C. Finally, staining with propidium iodide (PI) at 4°C was performed for 30 minutes in the dark, and cell fluorescence intensity was developed at 480 nm through flow cytometry (FACSCalibur flow cytometer, BD Biosciences) using Novoexpress version 1.0.2 software (ACEA Biosciences Inc.).
4
Cell Cycle Analysis of rAF-IL12-Treated HT29 Cells
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The rAF-IL12-treated HT29 cells (72-h post-infection) was subjected to cell cycle analysis using the Cycletest™ Plus DNA Reagent Kit (BD Biosciences, Franklin Lakes, NJ, USA). The cells pellet was first resuspended in solution A (trypsin), followed by in solution B (RNAse A) and finally stained with PI dyes, which requires a 10 mins of incubation period in each step. Later, the mixture was analysed by NovoCyte Flow Cytometer (ACEA Biosciences Inc., San Diego, CA, USA) using NovoExpress® version 1.2.4 software (ACEA Biosciences Inc., San Diego, CA, USA) (Syed Najmuddin et al., 2016 (link)).
5
Annexin V-FITC Apoptosis Assay
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The assay was carried out using Annexin V FITC Kit (BD Pharmigen, Franklin Lakes, NJ, USA) according to manufacturer’s protocol to evaluate the apoptosis induction of rAF-IL12 towards cancer cells. Briefly, the seeded cells (2 x 105 cells/well) were infected for 72 h before the cell pellets were harvested and resuspended in the 1X Binding buffer prior to staining with FITC Annexin V and propidium iodide (PI) dyes as described by Najmuddin et al. [19 (link)]. The cell suspension was then allowed to stand in the dark at room temperature for 15 min before being analysed by NovoCyte Flow Cytometer (ACEA Biosciences Inc., San Diego, CA, USA) using NovoExpress® version 1.2.4 software (ACEA Biosciences Inc., San Diego, CA, USA).
6
Lactobacillus Colonization of Tonsillar Tissue
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Tonsillar tissues were colonized with 15 vaginal Lactobacillus strains (27 blocks per condition) at a starting inoculum of 104 CFU/mL. At day 3 after inoculation with bacteria, all tissue blocks were collected and digested with collagenase IV (5 mg/mL; Gibco BRL) for 30 min with agitation in a Thermomixer at 900 rpm at 37°C. Following digestion, tissue cells were filtered with 100 μm cell strainers (Corning) and washed with 50 mL of PBS. Cells were then suspended in 1 mL of PBS and stained with 1 μl of live/dead Fixable Viability Dye eFluor 450 (EF 450, Invitrogen) for 15 min. After incubation, cells were washed and diluted in staining buffer [PBS, 1% normal mouse serum, 1% normal goat serum, 1 mM ethylenediaminetetraacetic acid (EDTA)] and stained with anti-CD3-allophycocyanin (CD3-APC) for 20 min. After surface staining, cells were permeabilized with the Fix&Perm Cell Fixation and Cell Permeabilization Kit (Invitrogen) then stained for 20 min with anti-Bcl2-PE, a mitochondrial anti-apoptotic antigen. Data were acquired with a Novocyte flow cytometer (ACEA Biosciences, CA, United States) equipped with 405, 488, and 640 nm laser lines using NovoExpress version 1.2.4 software (ACEA Biosciences) and analyzed using the same software.
7
Apoptosis Assay for HCT-116 and SW620 Cells
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HCT-116 and SW620 cells were plated in six-well plates at an initial density of 2.4x105 cells/well for 8 h in complete RPMI-1640 medium. Cells were divided into the following three treatment groups: i) DMSO (Control); ii) HT treatment (5, 10 and 20 µM in HCT-116 cells; 15, 30 and 60 µM in SW620 cells); and iii) a combination of HT and 3 mM NAC. Cells were starved in serum-free RPMI-1640 medium for 16 h, except that groups with NAC treatment were pretreated with 3 mM NAC for 2 h after starvation in serum-free medium for 14 h, followed by HT treatment for 24 h. Cells were harvested, washed twice with PBS (Thermo Fisher Scientific, Inc.), evaluated for apoptosis using FITC Annexin V Apoptosis Detection kit I according to the manufacturer's protocol and analyzed using Novocyte® flow cytometer with NovoExpress® version 1.2.4 software (ACEA Bioscience, Inc.).
8
Cell Cycle Analysis of CT26 Cancer Cells
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The CT26 cancer cells were seeded in 6-well plates at a concentration of 2x105 cells/well. After 72 h of treatment, the cells were centrifuged at 2500 x g speed for 5 min to obtain the cells pellet. The cells pellet was then, subjected to cell cycle analysis using CycletestTM Plus DNA Reagent Kit (BD Biosciences, Franklin Lakes, NJ, USA), as described by Najmuddin et al. [19 (link)]. The cells pellet was resuspended in the solution A (trypsin) and incubated for 10 min before solution B (RNAse A) was added. At this stage, a further 10 min was required. Finally, solution C (propidium iodide (PI) stain) was added to the mixture. After 10 min of incubation, the mixture was analysed by NovoCyte Flow Cytometer (ACEA Biosciences Inc., San Diego, CA, USA) using NovoExpress® version 1.2.4 software (ACEA Biosciences Inc., San Diego, CA, USA).
9
ROS Quantification in Colorectal Cells
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Cellular ROS content was measured by flow cytometry and fluorescence microscopy for quantitative and qualitative evaluation. HCT-116 or SW620 cells were plated in six-well plates at a density of 2.0x105 cells/well and allowed to attach overnight, and were then exposed to the indicated concentrations of HT for 4 h (5, 10 and 20 µM for HCT-116 cells; 15, 30 and 60 µM for SW620 cells). Cells were stained with 10 µM 2',7'-dichlorofluorescein diacetate (DCFH-DA; Beyotime Institute of Biotechnology) at 37˚C for 30 min and then washed three times in serum-free culture medium. For flow cytometry, cells were collected, and fluorescence was analyzed at excitation and emission wavelengths of 488 and 525 nm, respectively, using the Novocyte® flow cytometer with NovoExpress® version 1.2.4 software (ACEA Bioscience, Inc.). The mean value of DCFH-DA fluorescence intensity was utilized for quantitative analysis. For fluorescence microscopy, cell images were captured using the EVOS FL Imaging System (Thermo Fisher Scientific, Inc.).
10
Apoptosis Induction in Cancer Cells
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The assay was carried out using Annexin V FITC Kit (BD Pharmigen, San Diego, CA, USA) according to manufacturer’s protocol to evaluate the apoptosis induction of rAF-IL12 towards cancer cells. The pellets of rAF-IL12-treated HT29 cells (72-h post-infection) were resuspended in 1x Binding Buffer prior to staining with FITC Annexin V and propidium iodide (PI) dyes. The cell suspension was then allowed to stand in the dark at room temperature for 15 mins before being analysed using the NovoCyte Flow Cytometer (ACEA Biosciences Inc., San Diego, CA, USA) and the NovoExpress® version 1.2.4 software (ACEA Biosciences Inc., San Diego, CA, USA) (Pawłowska et al., 2018 (link)).
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