To detect macrophage surface markers, anti-CD163-PE (BD Biosciences, USA) and anti-CD11b-APC (eBioscience, USA) antibodies were used to stain cells at 4 °C for 30 min. Isotype controls were run in parallel. After washing, a BD Accuri C6 flow cytometer (BD Biosciences) was used to determine the proportion of CD11b+CD163+ macrophages. To detect cell cycle arrest, glioma cells were collected 48 h after transfection and fixed with ice-cold 70% ethanol overnight. Then, the cells were incubated with 0.5 mL of propidium iodide (BD Biosciences) in the dark at room temperature for 15 min and were finally analyzed with a BD Accuri C6 flow cytometer (BD Biosciences) and ModFit software (Verity Software House, USA). An Annexin V-FITC apoptosis detection kit (BD Biosciences) was utilized to evaluate apoptosis according to the manufacturer’s instructions. In brief, cells were stained with Annexin V-FITC and propidium iodide. After incubation at room temperature in the dark for 15 min, apoptosis was quantified using a BD Accuri C6 flow cytometer (BD Biosciences) within 1 h of staining.
Accuri c6 flow cytometer
Manufactured by BD
Sourced in United States, United Kingdom, Germany, Belgium, Canada, France, Italy, China, Switzerland, Australia, Japan, Brazil, Singapore
The BD Accuri C6 flow cytometer is a compact, automated instrument designed for cell analysis. It uses flow cytometry technology to precisely count and characterize cells in a sample. The BD Accuri C6 measures various parameters, such as cell size and granularity, to provide detailed information about the sample.
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Lab products found in correlation
Accuri c6, by BD (157 mentions)
Propidium iodide, by Merck Group (89 mentions)
Cflow plus software, by BD (46 mentions)
Fitc annexin 5 apoptosis detection kit, by BD (36 mentions)
Propidium iodide, by Thermo Fisher Scientific (33 mentions)
Fitc annexin 5 apoptosis detection kit 1, by BD (32 mentions)
Cflow software, by BD (30 mentions)
Propidium iodide, by BD (27 mentions)
Trypsin edta, by Thermo Fisher Scientific (26 mentions)
Rnase a, by Merck Group (23 mentions)
Triton x 100, by Merck Group (22 mentions)
Mitosox red, by Thermo Fisher Scientific (21 mentions)
Annexin 5 fitc, by BD (20 mentions)
Cellquest software, by BD (17 mentions)
Sybr green 1, by Thermo Fisher Scientific (17 mentions)
Bovine serum albumin (bsa), by Merck Group (17 mentions)
Cflow plus, by BD (15 mentions)
Trypsin, by Thermo Fisher Scientific (14 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (14 mentions)
Cycletest plus dna reagent kit, by BD (13 mentions)
3 652 protocols using accuri c6 flow cytometer
1
Macrophage Immunophenotyping and Cell Cycle Analysis
2
Apoptosis and Surface Marker Analysis
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The Annexin V-FITC Apoptosis Detection Kit was applied according to the manufacturer’s instructions (Merck). Briefly, Jurkat cells were collected following cell death induction and resuspended in Annexin V staining buffer for 30 min. PI (final concentration: 0.6 µg/mL) was added to each sample prior to analysis. Samples were analyzed using the BD Accuri™ C6 flow cytometer operating with BD Accuri™ C6 software. Additionally, flow cytometric analysis was performed on cells stained with antibodies against PS. To this end, cells were resuspended in PBS and stained with 0.2 µg/mL anti-PS-Alexa Fluor 488 antibody or the isotope control antibody, mouse IgG Alexa Fluor 488 (both from Merck Millipore) for 30 min. Flow cytometry was performed using the BD Accuri™ C6 flow cytometer and BD Accuri™ C6 software. Surface expression of CD31 was also determined by using flow cytometry. Following cell death induction, cells were stained with 1 µg/mL of the mouse anti-human CD31-FITC antibody (Bio-Rad Laboratories) or the FITC-labeled IgG1 isotope control antibody (BD Biosciences) and analyzed on a BD Accuri™ C6 flow cytometer.
3
Apoptosis and ROS Quantification by Flow Cytometry
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AnnexinV-647 and PI detection kit (Yeasen, Shanghai, China) was used to detect cell death. Cells were collected and washed with PBS and then resuspended in 1× binding buffer. Cells suspension (100 μL) was then transferred to a 5-mL culture tube and stained with 5 μL Annexin V-647 and 10 μL propidium iodide (PI) for 15 min at room temperature in dark. After addition of 200 μL binding buffer into each tube, the apoptotic cells were quantified using Accuri C6 flow cytometer (BD Biosciences, East Rutherford, NJ, USA). Cell Cycle Analysis Kit (Yeasen, China) was used to analyze DNA content and cell cycle profile. The cells were collected and washed with PBS and then placed in 70% ethanol overnight. Cell suspension (100 μL) was then transferred to a 5-mL culture tube and stained with 10 μL PI and 10 μL RNase for 30 min at room temperature in dark. After addition of 200 μL binding buffer into each tube, the apoptotic cells were quantified using Accuri C6 flow cytometer (BD Biosciences, East Rutherford, NJ, USA). Intracellular ROS generation was estimated using DCFH-DA (Yeasen, China). After cells were incubated with 10 μM DCFH-DA for 30 min and washed with PBS, measurements were performed using Accuri C6 flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). The ROS level is proportional to the mean fluorescence intensity (MFI) of a fluorescent probe DCFH-DA.
4
Quantifying Bacterial Adherence on Membranes
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P. aeruginosa PAO1 was incubated in an LB broth (BD, USA) for 16 h. The cultured cells were collected by centrifugation at 8000 rpm for 10 min and subsequently washed 2 times with a PBS buffer. A total of 2.5% of glutaraldehyde was added to the washed cells (OD600, 1.0) and incubated for 2 h, at RT, following the protocol described in [24 (link)]. Cells were washed 2 times with a PBS buffer, and the total number of cells was determined using a BD AccuriTM C6 flow cytometer (BD, USA).
The organic substance-conditioned RO membrane coupons (2 × 2 cm2) were placed in the 6-well microplate, to which we added 8 mL of PBS. Then, 8 µL of glutaraldehyde-treated or untreated P. aeruginosa PAO1 bacterial cells were injected into the conditioned membranes and placed in a 6-well microplate. The microplate was subsequently incubated at 30 °C and 90 rpm and took suspension samples at 0 h and 6 h. Total cell numbers were measured using BD AccuriTM C6 flow cytometer (BD, USA).
The organic substance-conditioned RO membrane coupons (2 × 2 cm2) were placed in the 6-well microplate, to which we added 8 mL of PBS. Then, 8 µL of glutaraldehyde-treated or untreated P. aeruginosa PAO1 bacterial cells were injected into the conditioned membranes and placed in a 6-well microplate. The microplate was subsequently incubated at 30 °C and 90 rpm and took suspension samples at 0 h and 6 h. Total cell numbers were measured using BD AccuriTM C6 flow cytometer (BD, USA).
5
Investigating JD's Cell Cycle Effects
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To determine the effect of JD on the cell cycle, 5.0×106 EC109/Taxol cells in the exponential growth phase were treated with various concentrations of JD for 24 h. After an incubation period, the cells were collected, centrifuged and fixed with ice-cold 70% ethanol at 4°C overnight. The cells were washed with PBS before staining with PI and then suspended in staining buffer (50 µg/ml PI, 1% Triton X-100 and 0.5 mg/ml RNase A in PBS) at 37°C for 30 min in the dark. The cells were analyzed on an Accuri C6 flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA). The histograms of DNA distribution were shown as a sum of cell populations at the G0/G1, G2/M or S phase using FlowJo software.
An Annexin V-fluorescein isothiocyanate kit (Annexin V- FITC; Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) was used to stain and identify the percentage of apoptotic cells. Briefly, the cells were seeded into 6-well plates and treated with 0, 10, 20 and 40 µM of JD for 24 h. The cells were then harvested, washed with ice-cold PBS and resuspended in binding buffer containing Annexin V-FITC (0.5 mg/ml) and PI (0.5 mg/ml). Following 15 min of incubation at room temperature in the dark, the stained cells were analyzed immediately by an Accuri C6 flow cytometer (Becton-Dickinson). Apoptotic cells were determined by cells that were both Annexin V-positive and PI-negative.
An Annexin V-fluorescein isothiocyanate kit (Annexin V- FITC; Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) was used to stain and identify the percentage of apoptotic cells. Briefly, the cells were seeded into 6-well plates and treated with 0, 10, 20 and 40 µM of JD for 24 h. The cells were then harvested, washed with ice-cold PBS and resuspended in binding buffer containing Annexin V-FITC (0.5 mg/ml) and PI (0.5 mg/ml). Following 15 min of incubation at room temperature in the dark, the stained cells were analyzed immediately by an Accuri C6 flow cytometer (Becton-Dickinson). Apoptotic cells were determined by cells that were both Annexin V-positive and PI-negative.
6
Apoptosis and Cell Cycle Analysis
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Cells were washed with PBS, then stained (106 cells) in buffer containing FITC-annexin V and 7-amino-actinomycin D (7-AAD) (Beckmann Coulter, Fullerton, CA, USA) for 15 min at 4 °C and analyzed by flow cytometry (Becton Dickinson Accuri™ C6 flow cytometer). For cell cycle analysis [45 (link)], cells were first incubated with fixing solution (PFA 2%, Hepes 1%, saponin 0.03%) for 15 min and then in PFS permeabilization solution (PBS 1×, SVF 10%, saponin 0.03%, Hepes 1%). Cells were next stained for 30 min at room temperature with anti-Ki67-Alexa Fluor 488 monoclonal antibody or the corresponding isotype as control (Becton-Dickinson, Franklin Lakes, NJ, USA) before analysis by flow cytometry (Becton Dickinson Accuri™ C6 flow cytometer). The FlowJo® software (V10.1, BD Biosciences, Franklin Lakes, NJ, USA) was used to analyze data.
7
Monitoring Grumpy Function in Trypanosoma brucei
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Cells were diluted at 5 × 104 parasites/ml and induced with tetracycline (1 μg/ml) for 3 days for grumpy RNAi and Tb927.10.12080 overexpression and for 6 days for grumpy overexpression cell lines. Cells were counted every day, live/dead cells were assessed by propidium iodide staining, GFP::PAD1-positive cells were scored, and all these parameters were quantified using an Accuri C6 flow cytometer. For grumpy RNAi/overexpression, at day 2 after tetracycline induction, RNA samples were collected by centrifugation of equivalent number of cells and addition of TRIzol reagent (Invitrogen) to the cell pellets. For the grumpy overexpression cell line, at day 3 after tetracycline induction, an equivalent number of cells were collected by centrifugation, and the cell cycle profiles were assessed using an Accuri C6 flow cytometer and propidium iodide staining in fixed cells.
8
Apoptosis Detection by Annexin V/7AAD and Caspase Assays
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Annexin V/7AAD double-staining [45 (link),46 (link)] and caspases 3, 8, and 9 activation assays [47 (link)] were used to detect apoptosis status. Annexin V-FITC/7AAD (1:1000/1 μg/mL) [48 (link)] (Strong Biotech; Taipei, Taiwan) was added to cells for 1 h incubation, and their fluorescence intensities were detected by Accuri C6 flow cytometer. The cell spots for annexin V (+)/7AAD (+ or −) intensity were counted as apoptosis (+) cells.
Moreover, the activities of caspases 3, 8, and 9 were detected by OncoImmunin’s specific peptides (Gaithersburg, MD, USA) according to the manufacturer’s instructions [47 (link),49 (link)]. When caspases 3, 8, and 9 are activated, these peptides generate fluorescence and are detected by the Accuri C6 flow cytometer.
Moreover, the activities of caspases 3, 8, and 9 were detected by OncoImmunin’s specific peptides (Gaithersburg, MD, USA) according to the manufacturer’s instructions [47 (link),49 (link)]. When caspases 3, 8, and 9 are activated, these peptides generate fluorescence and are detected by the Accuri C6 flow cytometer.
9
Quantifying TLR4 Binding via Yeast Display
10
Quantifying UV-Induced Cell Survival and Death
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Acute cell survival was determined using methylthiazolyldiphenyl‐tetrazolium bromide (MTT), which was added to cell culture medium at a final concentration of 0.25 mg ml−1 and incubated for 30 min before solubilization with DMSO and measurement of absorbance at 570 nm on a Synergy H1 spectrophotometer (Bio‐Tek). Absorbance values for the UV‐irradiated samples were normalized to the nonirradiated samples. Measurements of cell death involved addition of propidium iodide (25 µg ml−1) to unfixed suspensions of trypsinized cells harvested 24 h after UV exposure. The percentage of PI‐positive (dead) cells was determined using an Accuri C6 flow cytometer. To determine cell cycle distribution, ethanol‐fixed cells were stained with 25 µg ml−1 propidium iodide after RNase‐treatment and analyzed for DNA content using an Accuri C6 flow cytometer.
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