Facscalibur

Manufactured by BD

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The FACSCalibur is a flow cytometry system designed for multi-parameter analysis of cells and other particles. It features a blue (488 nm) and a red (635 nm) laser for excitation of fluorescent dyes. The instrument is capable of detecting forward scatter, side scatter, and up to four fluorescent parameters simultaneously.

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FACSCalibur machine (187 citations) FACSCalibur ow cytometer (147 citations) FACSCalibur flow (29 citations) FACSCalibur cell analyser (11 citations) BD FACSCalibur four-color analysis cytometer (10 citations) FACSCalibur laser flow cytometric system (2 citations) FACSCalibur laser cytometer (2 citations) FACSCalibur Benchtop Analyzer (2 citations) FACSCalibur flow meter (2 citations) Single-laser FACScalibur cytometer (1 citations)

18 538 protocols using facscalibur

Apoptosis and Cell Cycle Analysis

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For the apoptosis assays, annexin V staining was performed using the Annexin V-FITC Apoptosis Detection Kit (BD Biosciences, CA, USA) according to the manufacturer’s recommendations. The samples were examined by flow cytometry (FACS Calibur, BD, USA), and the data were analyzed with CELLQuest software (FACS Calibur, BD, USA).
For the cell cycle assays, following incubation, the cells were harvested, washed three times with PBS, centrifuged and fixed with 70% anhydrous ethanol overnight at 4 °C. They were then incubated with RNAase for 30 min and stained with PI for 15 min at 37 °C in the dark. The samples were examined by flow cytometry (FACS Calibur, BD, USA), and the data were analyzed with ModFit LT software (FACS Calibur, BD, USA).

Liu W., Wang X., Mei Z., Gong J., Huang L., Gao X., Zhao Y., Ma J, & Qian L. (2017). BNIP3L promotes cardiac fibrosis in cardiac fibroblasts through [Ca2+]i-TGF-β-Smad2/3 pathway. Scientific Reports, 7, 1906.

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Quantifying Apoptosis and Proliferation by Flow Cytometry

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To assess SFN induced cell death, cells were treated with indicated SFN concentrations for 24 h or with 1 μM staurosporine (Sigma-Aldrich) overnight, collected in FACS buffer (1x PBS with 2% FBS and 5 mM EDTA) and stained with 5 μg/ml propidium iodide (Sigma-Aldrich) and Annexin V-FITC (BD Bioscience). Red fluorescence intensity (propidium iodide) or green fluorescence intensity (Annexin V-FITC) of 10,000 individual cells was measured at a FACSCalibur (BD Bioscience). Cell debris was excluded from the analysis by gating on forward and side scatter.
For quantification of the apoptotic sub-G1 cells, cells were either left untreated or treated with 5 μM SFN for 24 h, collected, fixed in cold 80% ethanol at 4°C and stained with propidium iodide (5 μg/ml propidium iodide and 0.4 mg/ml RNase A in PBS). Red fluorescence intensity (propidium iodide) of 10,000 individual cells was measured at a FACSCalibur (BD Bioscience).
To assess cell proliferation, cells were pulse labeled by incubation with 5 μM CFSE (Sigma-Aldrich) for 20 min and either directly measured after the pulse or grown for 72 h in the presence of different SFN concentrations or in serum free medium (starvation). Green fluorescence intensity (CFSE) of 10,000 individual cells was measured at a FACSCalibur (BD Bioscience). Cell debris was excluded from the analysis by gating on forward and side scatter.

Bernkopf D.B., Daum G., Brückner M, & Behrens J. (2018). Sulforaphane inhibits growth and blocks Wnt/β-catenin signaling of colorectal cancer cells. Oncotarget, 9(74), 33982-33994.

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Apoptosis, Receptor Expression, and ROS Measurement

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The apoptotic rate was measured using an annexin V/PI assay and then analyzed with a flow cytometer (FACSCalibur, BD Biosciences). To measure receptor expression, a total of 1 × 10⁶ cells was resuspended in PBS and then incubated with PE-conjugated antibodies at room temperature for 0.5 h. human IgGs with PE were used as isotype controls. Cell fluorescence intensity was measured by flow cytometry (FACSCalibur, BD Biosciences), and the results were analyzed using FlowJo software. To measure intracellular reactive oxygen species (ROS) generation, the cells were pretreated with dichlorofluorescein diacetate (DCF-DA) (Sigma) at 37°C for 10 min before incubating with alternol for an additional 6 h. Cells were then harvested and washed with PBS, and the fluorescence intensity of intracellular DCF (excitation 488 nm, emission 530 nm) was monitored using a flow cytometer (FACSCalibur, BD Biosciences).

Ren Y., Wang X., Huang S., Xu Y., Weng G, & Yu R. (2021). Alternol Sensitizes Renal Carcinoma Cells to TRAIL-Induced Apoptosis. Frontiers in Pharmacology, 12, 560903.

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Cytokine-Induced Killer Cells Modulate Drug Resistance

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The cells were cultured in a 6-well plate for 24 h, then incubated with different ratios of CIK (10:1 or 20:1) for 72 h. The cells were then digested, resuspended, incubated with P-gp antibodies for 30 min at 4°C and washed twice in PBS. The fluorescence intensity of fluorescein isothiocyanate (FITC)-P-gp (Abcam, Burlingame, CA, USA) was analyzed by flow cytometry (FACSCalibur; BD Biosciences, Franklin Lakes, NJ, USA) at 488 nm.
The cells were cultured in a 6-well plate for 24 h, then incubated with different ratios of CIK for 72 h. The cells were then digested, resuspended, incubated with 10 μM Rh-123 (Sigma-Aldrich, San Francisco, CA, USA) for 60 min and washed twice in PBS. The fluorescence intensity of Rh-123 was analyzed by flow cytometry (FACSCalibur; BD Biosciences) at 488 nm.
The cells were cultured in a 6-well plate for 24 h, then incubated with different ratios of CIK (10:1 or 20:1) for 72 h. A total of 10 μg/ml ADR was added and co-cultured for 24 h, then the cells were digested, resuspended, incubated with Annexin V-FITC and propidium iodide (PI) for 15 min at 37°C and washed twice in PBS. The apoptosis rate was analyzed by flow cytometry (FACSCalibur; BD Biosciences) at 488 nm.

WANG L., DENG Q., WANG J., BAI X., XIAO X., LV H.R., ZHAO M.F, & LIU P.J. (2014). Effect of CIK on multidrug-resistance reversal and increasing the sensitivity of ADR in K562/ADR cells. Oncology Letters, 8(4), 1778-1782.

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Flow Cytometric Analysis of FKB-Induced Cell Cycle, Apoptosis, and Mitochondrial Changes in HeLa Cells

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Flow cytometry was used to evaluate the cell cycle profile, apoptosis, and mitochondrial membrane potential of FKB-treated HeLa cells. The cell cycle profile of untreated controls and HeLa cells treated with FKB for 48 hours was evaluated using BD Cycletest^TM Plus DNA kit according to the manufacturer’s protocol. Briefly, harvested control and treated HeLa cells were incubated with 250 µL of Solution A (10 minutes), followed by 200 µL of trypsin inhibitor and RNase buffer Solution B (10 minutes), and finally by 200 µL of propidium iodide (PI; 10 minutes). The samples were then analyzed by BD FACSCalibur using BD CellQuest Pro software (BD, USA). For apoptosis quantification, BD Annexin-V/PI apoptosis kit (BD, USA) was used according to the manufacturer’s protocol. In brief, harvested control and FKB-treated HeLa cells were incubated with 5 µL of AnnexinV-FITC and 5 µL of PI for 15 minutes. Then, the samples were topped up with 400 µL of binding buffer and analyzed by BD FACSCalibur using BD CellQuest Pro software (BD, USA). BD MitoScreen JC-1 kit was used for quantification of mitochondrial membrane potential. In brief, harvested control and FKB-treated HeLa cells were incubated with 500 µL of JC-1 working solution (15 minutes), washed, and resuspended with 500 µL of assay buffer. The samples were then analyzed by BD FACS Calibur using BD CellQuest Pro software (BD, USA).

Yeap S.K., Abu N., Akthar N., Ho W.Y., Ky H., Tan S.W., Alitheen N.B, & Kamarul T. (2016). Gene Expression Analysis Reveals the Concurrent Activation of Proapoptotic and Antioxidant-Defensive Mechanisms in Flavokawain B–Treated Cervical Cancer HeLa Cells. Integrative Cancer Therapies, 16(3), 373-384.

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Flow Cytometry Analysis of Pluripotency Markers

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Cells were rinsed once with PBS and detached from the plate using Accutase. Cells were centrifuged for 5 minutes at 200×g before being resuspended well in 4% paraformaldehyde (PFA) and fixed for 15 minutes. Subsequently, cells were rinsed twice in PBS before analyses on a BD FACSCalibur flow cytometer for GFP expression. Representative flow plots can be found in Figure S4B. To sort for eGFP⁺ and GFP^– populations, cells were trypsinized, washed in PBS and sorted directly into TRIzol (Life Technologies) using a BD FACS Aria. To determine endogenous OCT4 expression, cells were fixed as above, permeabilized in 0.5% triton-X and stained overnight at 4° with rabbit anti-human OCT4 antibody (1∶100; Cell Signaling). The next morning cells were rinsed three times in PBS before incubation with secondary antibody anti-rabbit FITC (1∶250; Jackson ImmunoResearch) for 1 hour at room temperature. Cells were analyzed on a BD FACSCalibur using appropriate unstained and secondary antibody only controls. Representative flow plots for OCT4 analysis can be found in Figure S4A. To determine CXCR4+ cells, cells were fixed in 4% PFA for 15 minutes, washed three times in PBS and incubated with CXCR4-PE antibody (1∶20; R&D) for 45 minutes. After cells were washed well, they were analyzed using BD FACSCalibur.

Krentz N.A., Nian C, & Lynn F.C. (2014). TALEN/CRISPR-Mediated eGFP Knock-In Add-On at the OCT4 Locus Does Not Impact Differentiation of Human Embryonic Stem Cells towards Endoderm. PLoS ONE, 9(12), e114275.

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Flow Cytometry Analysis of Receptor and Adaptor Expression

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Cells were analysed for receptor surface expression by flow cytometry using anti-Strep-tag II antibody Oyster 645 (IBA Lifesciences 2-1555-050) or anti-Strep-tag II antibody (IBA Lifesciences 2-1507-001) and anti-mouse IgG1 antibody Alexa Fluor 647 (Thermo Fisher Scientific A-21240) (BD FACSCalibur, 640-nm laser, FL4–661/16 band-pass filter, BD Biosciences). Introduced adaptor expression was tested via expression of EmGFP encoded on the adaptor lentivector (BD FACSCalibur, 488-nm laser, FL1–530/30 band-pass filter, BD Biosciences). Expression of 1G4 TCRβ and CD8α was analysed using anti-TCR Vβ 13.1 antibody FITC (H131; Thermo Fisher Scientific) and anti-CD8α antibody PE (HIT8a; Biolegend), respectively (BD FACSCalibur, 488-nm laser, FL1–530/30 band-pass filter and FL2–585/42 band-pass filter, BD Biosciences). Cells were sorted for high expression of receptor, introduced adaptor, or CD8 coreceptor by FACS (MoFlo Astrios, Beckman Coulter).

Denham E.M., Barton M.I., Black S.M., Bridge M.J., de Wet B., Paterson R.L., van der Merwe P.A, & Goyette J. (2019). A generic cell surface ligand system for studying cell–cell recognition. PLoS Biology, 17(12), e3000549.

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Cell Cycle and Apoptosis Analysis

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For cell cycle analysis, one million cells were centrifuged at 1030 g for 5 min, followed by resuspension in 100 µl of PBS at 4°C, and cells fixed by the addition of 100 µl of 4% PFA/PBS during 15 min on ice. After fixation, cells were centrifuged at 1030 g for 6 min at 4°C, and the cell pellet resuspended in 340 µl PBS, followed by incubation with RNAse A (Roche; 10 109 169 001) (3 µl of 10 mg/ml stock) and propidium iodide (Sigma–Aldrich; P4864; 1 mg/ml) (12 µl of 1 mg/ml stock) for 20 min at 37°C in the dark. Flow cytometry analysis was performed in a FACScalibur^TM (Becton Dickinson), propidium iodide-positive signals analyzed using a 670 nm fluorescence emission filter, and data represented with respect to the amount of DNA present per cell.
Analysis of apoptosis was performed using Alexa Fluor-488 AnnexinV/Dead Cell Apoptosis Kit (Invitrogen, V13241) according to the manufacturer's instructions. Briefly, one million cells were centrifuged at 500 g for 3 min, and the pellet washed with 1 ml of ice-cold PBS. Cells (0.5 × 10⁶) were resuspended in 100 µl of binding buffer and labeled with AlexaFluor 488-AnnexinV and propidium iodide for 15 min on ice. Cells were subsequently analyzed by flow cytometry in a FACScalibur^TM (Becton Dickinson) using a 488 nm excitation laser and emission at 530 nm (annexinV) and 670 nm (propidium iodide).

Fernández B., Lara Ordóñez A.J., Fdez E., Mutez E., Comptdaer T., Leghay C., Kreisler A., Simonin C., Vandewynckel L., Defebvre L., Destée A., Bleuse S., Taymans J.M., Chartier-Harlin M.C, & Hilfiker S. (2019). Centrosomal cohesion deficits as cellular biomarker in lymphoblastoid cell lines from LRRK2 Parkinson's disease patients. Biochemical Journal, 476(19), 2797-2813.

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Assessing Cell Viability and P2X7 Uptake

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Cell viability was assessed by annexin V/propidium iodide staining (eBioscience, San Diego, CA, USA). Briefly, cells were harvested with trypsin-EDTA, suspended in staining buffer, stained for 20 min with annexin V and propidium iodide, and analyzed using a BD FACSCalibur TM and BD CellQuest TM Pro (Becton, Dickinson Biosciences, San Jose, CA, USA).
P2X 7 channel uptake YO-PRO-1, a 629-Da fluorescent nucleic acid dye that is transported across the cell membrane by P2X 7 , was used to quantify the uptake of extracellular ATP, as previously described (Cankurtaran-Sayar et al. 2009) . In brief, cells were collected with trypsin-EDTA, centrifuged at 1,500 rpm for 5 min, washed once with cold phosphatebuffered saline, and stained with 2 μM YO-PRO-1 (Invitrogen, Carlsbad, CA, USA) for 30 min in the dark. The mean fluorescence intensity was then analyzed using a BD FACSCalibur TM and BD CellQuest TM Pro (Becton, Dickinson Biosciences, San Jose, CA, USA).

Ho V.M., Hirohashi N., Kong W.S., Yun G., Ota K., Itai J., Yamaga S., Suzuki K., Tanigawa K., Kanno M, & Shime N. (2018). Sera from Septic Patients Contain the Inhibiting Activity of the Extracellular ATP-Dependent Inflammasome Pathway. The Tohoku journal of experimental medicine, 245(3).

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Flow Cytometric Analysis of Cell Death and ROS

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The fraction of dead cells was quantified by flow cytometry (FACS Calibur, Becton Dickinson, Heidelberg, Germany; FL-2) of PI–stained cells as described previously [6 (link),8 (link),9 (link)]. Cells were incubated for 30 min in the dark with PI (10 μg/mL, Thermo Scientific, Waltham, MA, USA) in PBS and measured within 1 h.
To quantify cellular ROS production, cells were stained for 30 min at 37 °C with 5 μM Dihydroethidium (DHE) in RPMI 1640 medium (Molecular Probes/Invitrogen, Carlsbad, CA, USA). DHE-positive cells were detected by flow cytometry (BD FACS Calibur, Becton Dickinson; FL-2). ROS-production was evaluated by quantification of the fraction of DHE-positive cells (at least 10,000) with higher fluorescence.

Hansel C., Hlouschek J., Xiang K., Melnikova M., Thomale J., Helleday T., Jendrossek V, & Matschke J. (2021). Adaptation to Chronic-Cycling Hypoxia Renders Cancer Cells Resistant to MTH1-Inhibitor Treatment Which Can Be Counteracted by Glutathione Depletion. Cells, 10(11), 3040.

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