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Cytoflex

Manufactured by Beckman Coulter
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The CytoFLEX is a flow cytometry system designed for a wide range of applications. It features a compact design and offers multi-color analysis capabilities.

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Lab products found in correlation

Cytexpert software, by Beckman Coulter (123 mentions) Cytexpert, by Beckman Coulter (65 mentions) Cytexpert 2, by Beckman Coulter (46 mentions) Propidium iodide, by Merck Group (31 mentions) Facscanto 2, by BD (29 mentions) Software, by FlowJo (27 mentions) Trypsin edta, by Thermo Fisher Scientific (22 mentions) Fitc annexin 5 apoptosis detection kit 1, by BD (21 mentions) Ionomycin, by Merck Group (18 mentions) Annexin 5 fitc apoptosis detection kit, by Beyotime (18 mentions) Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (18 mentions) Facscalibur, by BD (17 mentions) Accuri c6, by BD (17 mentions) Fitc annexin 5 apoptosis detection kit, by BD (16 mentions) Lsrfortessa, by BD (16 mentions) Mitosox red, by Thermo Fisher Scientific (16 mentions) Hoechst 33342, by Thermo Fisher Scientific (15 mentions) Facsaria 3, by BD (14 mentions) Attune nxt, by Thermo Fisher Scientific (14 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (13 mentions)

3 030 protocols using cytoflex

1

Cell Cycle and Apoptosis Analysis in Cell Lines

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The cell cycle was detected using the DNA content quantification assay (Solarbio, CA1510, Beijing, China). Briefly, cells were fixed with 75% alcohol overnight at 4 °C after digestion with trypsin (Gibco, 25200072, Waltham, MA, USA). RNase A was used the next day to remove RNA. Then PI staining solution was added, and the cells were incubated for 30 min in the dark at 4 °C. The cell cycle was finally detected using Beckman Coulter Cytoflex (BeckmanCoulter, Brea, CA, USA).
Apoptosis was detected using an Annexin V-FITC apoptosis detection kit (Solarbio, CA1020, Beijing, China). Briefly, cells were digested with an EDTA-free trypsin (Gibco, 15050057, Waltham, MA, USA) and resuspended in a 1× binding buffer. FITC-labeled anti-Annexin V was added for 5 min at room temperature in the dark. Finally, the PI staining solution was added, and cell apoptosis was detected using Beckman Coulter Cytoflex (BeckmanCoulter, Brea, CA, USA).
Chen Z., Bao H., Long J., Zhao P., Hu X., Wang H., Zhang Y., Yang J., Zhuge Q, & Xia L. (2023). GBE1 Promotes Glioma Progression by Enhancing Aerobic Glycolysis through Inhibition of FBP1. Cancers, 15(5), 1594.
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2

Mitochondrial Superoxide and Membrane Potential

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To measure the relative levels of mitochondrial superoxide, cells were resuspended in HBSS and stained with 5 μM MitoSOX Red (Invitrogen, M36008) for 10 min at 37 °C. Cells were then washed three times with HBSS. Using a flow cytometer (CytoFLEX, Beckman), MitoSOX Red was measured using the PE channel. About 10 μM CCCP was used as a control, which increases mitochondrial ROS. Relative fluorescence intensity from biological triplicates of 10,000 cells were used as an indicator of mitochondrial superoxide levels. To measure mitochondrial membrane potential, cells were stained with a culture medium with 1× JC-1 staining solution (YEASON, 40706ES60) for 20 min at 37 °C. Cells were then washed three times with the staining buffer and subjected to flow cytometry (CytoFLEX, Beckman Coulter) following the manufacturer’s instructions. Briefly, JC-1 was excited at 488 nm and its emission at both 525 nm (FITC-A) and 585 nm (PE-A) were measured. By comparing the ratios of emission at PE/FITC, relative levels of mitochondrial membrane potential were determined from the 5000 cells in the biological triplicate. Ten micromolar CCCP was used as control which decreased mitochondrial membrane potential.
Zhang Z., Zeng D., Zhang W., Chen A., Lei J., Liu F., Deng B., Zhuo J., He B., Yan M., Lei X., Wang S., Lam E.W., Liu Q, & Wang Z. (2021). Modulation of oxidative phosphorylation augments antineoplastic activity of mitotic aurora kinase inhibition. Cell Death & Disease, 12(10), 893.
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3

Yeast Cell Survival Assay with Extracts

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Yeast cells were grown in liquid medium until the mid-late exponential phase, centrifugated (13,000 rpm, 5 min), and then inoculated at 0.150 OD/mL (Optical Density/mL) in flasks containing medium in the absence (negative control) or presence of the extracts (0.2% concentration). Survival was assessed by propidium iodide staining (PI) at the indicated time points with a Cytoflex cytofluorimeter (Beckman Coulter, Milan, Italy) and analyzed with the Cytoflex software (version 2.6.0.105), as previously reported [28 (link),29 (link)].
Docimo T., Celano R., Lambiase A., Di Sanzo R., Serio S., Santoro V., Coccetti P., Russo M., Rastrelli L, & Piccinelli A.L. (2024). Exploring Influence of Production Area and Harvest Time on Specialized Metabolite Content of Glycyrrhiza glabra Leaves and Evaluation of Antioxidant and Anti-Aging Properties. Antioxidants, 13(1), 93.
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4

Apoptosis and Cell Cycle Analysis

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Flow cytometric analysis of cell apoptosis was detected by using the Annexin V-FITC/Propidium Iodide (PI) Apoptosis Detection Kit (LiankeBio, Hangzhou, China). CRC cells were plated in 6-well plates in complete medium. 24 h later, different indicated concentration of C.B CM or 5-FU were dissolved into fresh medium to replace the old medium. Cells were treated for 24 h, and then both supernatant and adherent cells were collected by trypsinization. The cell suspension used for analysis was prepared with 500 μL cold PBS containing around 1 × 106 (link) cells. Annexin V-FITC (10 μL) and PI (5 μL) were added, respectively, and stained at room temperature in the dark for 5 min. The pretreated cells were analyzed by flow cytometry (CytoFLEX, Beckman Coulter, Inc., Miami, FL, USA).
For cell cycle distribution analysis, a propidium iodide (PI) cell cycle staining kit (LiankeBio, Hangzhou, China) was used according to the manufacturer’s instructions. The cell cycle phases were determined by flow cytometry (CytoFLEX, Beckman Coulter, Inc., Miami, FL, USA).
Xu H., Luo H., Zhang J., Li K, & Lee M.H. (2023). Therapeutic potential of Clostridium butyricum anticancer effects in colorectal cancer. Gut Microbes, 15(1), 2186114.
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5

Apoptosis and Cell Cycle Analysis

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Cell apoptosis was detected by Annexin V‐FITC Apoptosis Detection Kit (KeyGEN BioTECH, China) following the manufacturer's instruction and then analysed with CytoFLEX (Beckman Coulter, Brea, CA, USA). Cell cycle assay was carried out using PI/RNase Staining Kit (Dojindo) and FITC BrdU Flow Kit (BD Pharmigen, San Diego, CA, USA) in accordance with the manufacturer's instruction. Flow cytometry analysis was performed using CytoFLEX (Beckman Coulter).
Chen Z., Zhu W., Zhu S., Sun K., Liao J., Liu H., Dai Z., Han H., Ren X., Yang Q., Zheng S., Peng B., Peng S., Kuang M, & Lin S. (2021). METTL1 promotes hepatocarcinogenesis via m7G tRNA modification‐dependent translation control. Clinical and Translational Medicine, 11(12), e661.
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6

OT-I CD8+ T Cell Cytotoxicity Assay

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OT-I CD8+ T cells were co-cultured with either tumor cells or tumor expressing H-2Kb-OVA257-264 cells for 2 and 48 h. In the 2 h test, cells were co-cultured at the presence of anti-CD107a (Biolegend, 1D4B). After 2h, all cells were collected and stained with anti-CD8a (Biolegend) for degranulation analysis via flow cytometry (Cytoflex, Beckman). After 48 h, all cells were collected and stained with anti-CD8a, PI and Annexin V (Biolegend) for target cell apoptosis analysis via flow cytometry (Cytoflex, Beckman). All FCM data were analyzed by Flowjo 10.
Lu Z., Ni K., Wang Y., Zhou Y., Li Y., Yan J., Song Q., Liu M., Xu Y., Yu Z., Guo T, & Ma L. (2022). An in-library ligation strategy and its application in CRISPR/Cas9 screening of high-order gRNA combinations. Nucleic Acids Research, 50(11), 6575-6586.
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7

Cell Cycle Analysis of Mesothelioma Cells

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Mesothelioma cells were treated with QV0 (IC25), and at 48 h post-treatment, the cells were harvested and washed three times with phosphate-buffered saline (PBS). The cells were subsequently fixed in 70% ethanol for at least 30 min. For cell cycle analysis, the fixing solution was removed, and cells were treated with 0.01% RNase (10 mg/ml, Sigma-Aldrich) and 0.05% propidium iodide (PI) (Sigma-Aldrich) in PBS for 30 min at 37°C in the dark. The cell cycle distribution was determined on a CytoFLEX flow cytometer (Beckman Coulter, Miami Lakes, FL) within 30 min. The flow cytometer was calibrated using calibration beads according to the manufacturer’s instructions (CytoFLEX, Beckman). The flow cytometer was routinely operated at the slow flow rate setting (μl sample/minute), and the data acquisition for a single sample typically took 3–5 min. For each sample, 10,000 events of single cells were counted, and the cell cycle was analyzed using FlowJo software (Ashland, OR, USA).
Shi H., Zhang L., Yu T.K., Zhuang L., Ke H., Johnson B., Rath E., Lee K., Klebe S., Kao S., Qin K.L., Pham H.N., Vuong Q, & Cheng Y.Y. (2023). Leptospermum extract (QV0) suppresses pleural mesothelioma tumor growth in vitro and in vivo by mitochondrial dysfunction associated apoptosis. Frontiers in Oncology, 13, 1162027.
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8

Evaluating MC3T3-E1 Apoptosis on Modified Ti

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To evaluate the early apoptosis of MC3T3-E1, we measured the mitochondrial membrane potential by the JC-1 probe. Briefly, cells cultured for 72 h on different modified Ti surfaces in vitro in high-glucose environment were incubated with the JC-1 staining solution (Beyotime, Shanghai, China) at 37°C for 20 min, and then rinsed twice with phosphate-buffered saline (PBS). The mitochondrial membrane potential was determined according to the manufacturer’s instructions. The fluorescence intensity was evaluated by flow cytometry (Cytoflex, Beckman, USA), and CellQuest™ software (CytExpert, USA) was used to analyze the red/green fluorescence intensity ratio.
To further detect cellular apoptosis on different modified Ti surfaces subjected to high-glucose conditions, we used an Annexin V/PI apoptosis kit (Beyotime, Shanghai, China). After 72-h culture in high-glucose concentration, MC3T3-E1 cells were harvested and washed with PBS, and then re-suspended in 195 µL binding buffer. Thereafter, the cells were incubated with 10 µL PI and 5 µL mixture of Annexin V and fluorescein isothiocyanate (FITC) for 20 min in the darkroom at 37°C, and then evaluated by flow cytometry (Cytoflex, Beckman, USA). Data analysis was conducted using the CellQuest™ software (CytExpert, USA).
Yang J., Zhang H., Chan S.M., Li R., Wu Y., Cai M., Wang A, & Wang Y. (2020). TiO2 Nanotubes Alleviate Diabetes-Induced Osteogenetic Inhibition. International Journal of Nanomedicine, 15, 3523-3537.
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9

Apoptosis and Cell Cycle Analysis

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Annexin V apoptosis assay was performed using the Roche Annexin-V-FLUOS staining Kit. Cells were collected after treatment and pelleted by centrifugation. After a PBS wash, the cells were resuspended in Annexin-V-FLUOS labelling solution with Annexin V-FITC and propidium iodide, and incubated in room temperature for 10 min. The cells were then analysed using flow cytometry (CytoFLEX, Beckman Coulter, Krefeld, Germany). Similarly, Nicoletti assay for cell cycle analysis was performed by permeabilising cells with a 50:50 mixture of 1% sodium citrate and 1% Triton X. Subsequently the cells were supplemented with 100 ng/mL of propidium iodide and RNase A, incubated overnight, followed by flow cytometry and analysis (CytoFLEX, Beckman Coulter).
Sankaranarayanan R.A., Peil J., Vogg A.T., Bolm C., Terhorst S., Classen A., Bauwens M., Maurer J., Mottaghy F, & Morgenroth A. (2022). Auger Emitter Conjugated PARP Inhibitor for Therapy in Triple Negative Breast Cancers: A Comparative In-Vitro Study. Cancers, 14(1), 230.
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10

Intracellular Staining of Splenocytes

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Tissues were disrupted by passing through a nylon mesh (Kyoshin Ricoh). After red blood cell lysis with ammonium chloride potassium buffer, cells were incubated with an anti-CD16/CD32 (2.4G2; BD Pharmingen) to reduce nonspecific labeling of the cells before staining. Single cells were stained with fluorophore-labeled antibodies. For intracellular staining, splenocytes were fixed and permeabilized with Intracellular Fixation and Permeabilization Buffer set (eBioscience) or Foxp3 Staining Buffer Set (eBioscience) before intracellular staining with PE-conjugated anti-TLR9, anti-IRF4, pS6, Alexa647-conjugated anti-IRF4, and then analyzed on Cytoflex (Beckman Coulter). The data were acquired on a Cytoflex (Beckman Coulter) and analyzed with FlowJo software (Tree Star).
Higuchi R., Tanaka K., Saito Y., Murakami D., Nakagawa T., Nutt S.L., Ohkawa Y, & Baba Y. (2024). Type I interferon promotes the fate of Toll-like receptor 9–stimulated follicular B cells to plasma cell differentiation. PNAS Nexus, 3(4), pgae152.
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