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Facscan instrument

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The FACScan instrument is a flow cytometry system designed for cell analysis and sorting. It is capable of detecting and measuring multiple parameters of individual cells or particles suspended in a fluid stream.

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Lab products found in correlation

Pi rnase staining kits, by MultiSciences Biotech (4 mentions) Cellquest software, by BD (3 mentions) Cellquest pro software, by BD (3 mentions) Annexin 5 fluorescein isothiocyanate fitc apoptosis detection kit, by Keygen Biotech (3 mentions) Fitc labeled annexin 5 and pi staining, by BD (2 mentions) Bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Cellquest program, by BD (2 mentions) Propidium iodide ribonuclease staining solution, by BD (2 mentions) Fitc annexin 5 apoptosis detection kit 1, by BD (2 mentions) Dimethyl sulfoxide (dmso), by Thermo Fisher Scientific (2 mentions) Fitc annexin 5 apoptosis detection kit, by BD (1 mentions) Apopercentage apoptosis assay, by Biocolor (1 mentions) Anti human antibodies against cd3, by BD (1 mentions) Gallios cytometer, by Beckman Coulter (1 mentions) Fc blocking reagent, by BD (1 mentions) Cd11b percp cy5, by BioLegend (1 mentions) Lipopolysaccharides (lps), by Merck Group (1 mentions) Phosphate buffered saline (pbs), by Merck Group (1 mentions) Kaluza software, by Beckman Coulter (1 mentions) Annexin 5 fluorescein isothiocyanate propidium iodide fitc pi apoptosis detection kit, by MultiSciences Biotech (1 mentions)

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FACScan (3082 citations)

44 protocols using facscan instrument

1

Characterizing Cell Purity, Cycle, and Markers

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To evaluate cell purity, cells were fixed for 15 min in 4% formaldehyde, permeabilized in precooled 90% methanol for 10 min, blocked in 10% goat serum, and then incubated with an anti-cytokeratin (pan)-fluorescein isothiocyanate antibody (4545; Cell Signaling Technology, CST) diluted with PBS supplemented with 0.5% bovine serum albumin (incubation buffer) for 1 h. The cells were incubated in Alexa Fluor®-conjugated secondary antibody diluted with incubation buffer for 30 min and then counted by flow cytometry on a FACScan instrument (BD Bioscience).
To analyze cell cycle distribution, cells in the exponential growth phase were processed according to the manufacturer’s instructions (Dojindo) and then sorted by flow cytometry.
To determine the expression of CD24/CD44, cells were suspended in 100 μl PBS supplemented with 10 μl PE mouse anti-human CD24 (555428; BD Biosciences) and FITC mouse anti-human CD44 (555478; BD Biosciences) antibodies. After incubation for 30 min at 4°C in the dark, the cells were examined by flow cytometry on a FACScan instrument (BD Bioscience).
Li Z., Zhuo W., Chen L., Zhang X., Chen C., Hu D., Chen Y., Yang J., Zhou Y., Mao M., Xu L., Ju S., Shen J., Wang Q., Dong M., Xie S., Zhou J, & Wang L. (2021). Establishment and Characterization of a HER2-Positive Cell Line Derived From the Pleural Effusion of a Drug-Resistant Breast Cancer Patient. Frontiers in Cell and Developmental Biology, 9, 680968.
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2

Proliferation and Apoptosis Assays

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Triplicate samples of 5,000 cells were seeded in 96-well plates in medium containing puromycin for 48 h before proliferation was assessed using CellTiter 96 (Promega) at 570 nm absorbance using a Synergy-2 multi-detection plate reader (BioTek). Alternatively, cells were incubated with 10 µM bromodeoxyuridine (BrdU) for 2 h, washed in PBS, and fixed in 70% ethanol. DNA was denatured in 2 N HCl at room temperature for 15 min and resuspended in 0.1 M Na2B4O7, followed by staining with FITC-conjugated BrdU Ab (BD Pharmingen), washing in PBS, additional staining with 10 mg/ml propidium iodide for 30 min, and analysis on a FACScan instrument (Becton-Dickinson). For analysis of apoptosis, we used a FITC Annexin V Apoptosis detection kit I (BD Pharmingen) and analyzed cells on a FACScan instrument (Becton-Dickinson). Phospho-Histone H3 was analyzed as described previously34 (link), using Histone H3 (Cell Signaling, cat. no. 9715) and Phospho-Histone H3 (Cell Signaling, 3377) Ab.
Cejas P., Cavazza A., Yandava C., Moreno V., Horst D., Moreno-Rubio J., Burgos E., Mendiola M., Taing L., Goel A., Feliu J, & Shivdasani R.A. (2016). Transcriptional regulator CNOT3 defines an aggressive colorectal cancer subtype. Cancer research, 77(3), 766-779.
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3

Determining FLS Apoptosis via H2O2

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The cell death rate of FLSs in response to H2O2 treatment was determined using the Annexin V/FITC Apoptosis Detection Kit (BD Biosciences, San Jose, CA, USA) according to the manufacturer's protocol. FLAs-RA or control FLSs (4 × 105 cells/well) were plated in 6-well plates for 24 h. After treatment, cells were trypsinized, washed with PBS, and incubated with Annexin V and propidium iodide (PI) for 40 min in the dark. Apoptosis rates were assessed by the FACScan instrument (BD Biosciences, San Jose, CA, USA). Experiments were performed in triplicate and repeated three times.
Zhang M., Lu N., Li H.J., Guo X.Y., Lu L, & Guo Y. (2022). Inhibition of lncRNA NEAT1 induces dysfunction of fibroblast-like synoviocytes in rheumatoid arthritis via miRNA-338-3p-mediated regulation of glutamine metabolism. Journal of Orthopaedic Surgery and Research, 17, 401.
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4

Assessing Cell Apoptosis via Annexin V-FITC/PI Staining

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Apoptosis was analyzed using an Annexin V-FITC/propidium iodide (PI) staining kit (cat. no. V13242; Invitrogen; Thermo Fisher Scientific, Inc.). Fibroblasts (2×105 cells/well) were cultured in 6-well plates for 24 h at 37°C, then washed with cold PBS and centrifuged at 1,000 × g (5 min, 4°C). The cells were subsequently resuspended in 1X Annexin binding buffer, 5 µl Annexin V-FITC and 5 µl PI was added to the cells. The cells were incubated at room temperature for 15 min, then added to 400 µl 1X Annexin binding buffer. Cell apoptosis was assessed by flow cytometry using a FACScan instrument (BD Biosciences) equipped with CellQuest software (version 5.1; BD Biosciences). The apoptosis rate was calculated as the sum of early-apoptotic and late-apoptotic ell frequencies. Unstained cells, as well as Annexin V and PI single-stained cells were used as controls, and the gates for positive staining were set according to these controls.
He X, & Dong Y. (2020). Ankylosis progressive homolog upregulation inhibits cell viability and mineralization during fibroblast ossification by regulating the Wnt/β-catenin signaling pathway. Molecular Medicine Reports, 22(6), 4551-4560.
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5

Apoptosis and Cell Cycle Analysis

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After drug treatment or transfection for 24 hours, wash the cells with pre‐cooled phosphate‐buffered saline (PBS). We used the Propidium Iodide (PI)/RNase staining kits (Thermo Fisher Scientific) to analyse cell cycle. And the apoptosis of experimental cells was detected by using annexin V‐fluorescein isothiocyanate (FITC) apoptosis detection kits (Abnova) in a FACScan instrument (BD Biosciences). What else, to better show the state of cells, we used a fluorescence microscope (IX71, Olympus) to photograph the morphological features of apoptotic cells after they were harvested and suspended in PBS containing fluorescence dye dual acridine orange/ethidium bromide (AO/EB) (AMRESCO) (Sigma‐Aldrich) and both AO and EB were at the concentration of 100 mg/L in PBS.
Li X, & Yu Q. (2019). PON1 hypermethylation is associated with progression of renal cell carcinoma. Journal of Cellular and Molecular Medicine, 23(10), 6646-6657.
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6

Quantification of Apoptosis in Cell Cultures

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The APOPercentage™ apoptosis assay (Biocolor Ltd., Carrickfergus, Ireland) was used to quantify the apoptosis in cell cultures treated with holamine or funtumine, following a protocol described by Meyer et al. [31 (link)]. The cells were seeded in 12-well cell culture plates at a density 1 × 105 cells/mL and were treated with 10 to 30 µg/mL of the compounds for 24 h. After treatment, the apoptotic cells were analyzed using a Becton Dickinson FACScan instrument (BD Biosciences Pharmingen, San Diego, CA, USA). A minimum of 10,000 events per sample were recorded. The data were analyzed using the CELLQuest PRO® software (BD Biosciences Pharmingen, San Diego, CA, USA).
Badmus J.A., Ekpo O.E., Sharma J.R., Sibuyi N.R., Meyer M., Hussein A.A, & Hiss D.C. (2020). An Insight into the Mechanism of Holamine- and Funtumine-Induced Cell Death in Cancer Cells. Molecules, 25(23), 5716.
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7

Immunophenotyping of CAR-T cells

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Expression of M28z and effLuc CAR was detected using GFP and Thy1.1 APC-conjugated F(ab)2 anti-human immunoglobulin G1 (IgG1), respectively. Anti-human antibodies against CD3 (BD Biosciences, San Jose, CA) were used. Stained cells were processed using a FACScan instrument (BD Biosciences), and data were analyzed using FlowJo software (version 6.0; TreeStar, Ashland, OR).
Skovgard M.S., Hocine H.R., Saini J.K., Moroz M., Bellis R.Y., Banerjee S., Morello A., Ponomarev V., Villena-Vargas J, & Adusumilli P.S. (2021). Imaging CAR T-cell kinetics in solid tumors: Translational implications. Molecular Therapy Oncolytics, 22, 355-367.
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8

Flow Cytometry Analysis of Myeloid Cells

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Single-cell suspension obtained from spleen, BM and PB isolated from NB-bearing and healthy animals were washed with PBS (Sigma), incubated with Fc blocking reagent (BD Biosciences, San Diego, CA, USA) and then stained for 20 min (min) at 4 °C with the relevant Ab. The Abs used were: CD11b PerCP/Cy5.5 (BioLegend, London, UK), Ly-6G/Ly-6C (Gr-1) APC (BioLegend) and isotype-matched control monoclonal antibodies (Biolegend). After staining, samples were acquired with a Gallios cytometer (Beckman Coulter, Milan, Italy) and data were analyzed using Kaluza software (Beckman Coulter).
In some experiments, MSC-1 and MSC-2 cell lines were pretreated or not with LPS (Sigma) (1 μg/ml) for 4 h and then treated with BzATP (100 and 300 μM) for 30 min. The cells were fixed with 2% paraformaldehyde at RT for 20 min and permeabilized with permeabilization buffer (PBS, 1% FBS, 0.1% saponin, Sigma). Then, cells (5 × 105/tube) were incubated with anti-ARG-1 polyclonal Ab from Sigma and Santa Cruz Biotechnology (Heidelbergh, Germany) for 30 min at RT, washed twice with permeabilization buffer and incubated with FITC-conjugated F(ab')2 fragments of goat anti-rabbit IgG antibodies (Abcam, Cambridge, UK). After resuspension in staining buffer, cells were then analyzed by flow cytometry using a FACScan instrument (BD Biosciences, San Josè, CA, USA).
Bianchi G., Vuerich M., Pellegatti P., Marimpietri D., Emionite L., Marigo I., Bronte V., Di Virgilio F., Pistoia V, & Raffaghello L. (2014). ATP/P2X7 axis modulates myeloid-derived suppressor cell functions in neuroblastoma microenvironment. Cell Death & Disease, 5(3), e1135-.
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9

Cell Cycle and Apoptosis Analysis

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Cells were harvested and washed with ice-cold phosphate-buffered saline (PBS). The PI/RNase staining kits (Multi-sciences, Shanghai, China) and an annexin V-fluorescein isothiocyanate propidium iodide (FITC/PI) apoptosis detection kit (Multisciences, Shanghai, China) were used to detect cell cycle and apoptosis according to the manufacturer’s protocol using a FACScan instrument (BD, Franklin Lakes, NJ, USA), respectively. Three independent experiments were performed.
Xu D., Chen Y., Yuan C., Zhang S, & Peng W. (2019). Long non-coding RNA LINC00662 promotes proliferation and migration in oral squamous cell carcinoma. OncoTargets and therapy, 12, 647-656.
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10

Protein and Phenotypic Analysis of Cell Lines

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CAF, MCF-10A, ZR-75-1, HCT116, QBC939, NCCIT, RBE, HUCCT1, ZJU-0826, and ZJU-1125 cells were washed thrice with ice-cold phosphate buffered saline (PBS), lysed in RIPA buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 0.5% sodium deoxyholate, 1% Nonide P-40, and 0.1% SDS with Pierce Protease Inhibitor Tablets (AEBSF, aprotinin, bestatin, E-64, leupeptin, and pepstain A) (Thermo Fisher Scientific Inc., Waltham, MA, USA) and separated by centrifugation. The protein concentration was detected using the BCA Protein Assay Kit (Thermo Fisher Scientific Inc). Antibodies against the following proteins were used: E-cadherin, N-cadherin, β-catenin, α-SMA, MUC1, CD146, SOX17, Vitamin D3 Receptor (VDR), pdx1, CD326, FoxA1/HNF3α, FoxA2/HNF3β, Nanog, GAPDH, and β-actin (all 1:1000, from Cell Signaling Technology, Danvers, MA, USA).
Flow cytometry was performed using a FACScan instrument (BD Bioscience, San Jose, CA, USA) and commercially available reagents for cells at passages 3–5 and 60–65. A panel of monoclonal antibodies was evaluated, including CD24, CD44, CD29, CD34, CD90, CD117, CD133, CD184, CD326, and CD338 (Biolegend, San Diego, CA, USA). Antigen expression was determined based on a significant shift in staining compared to an isotype control.
Zhang Y., Luo J., Dong X., Yang F., Zhang M., Zhao J., Wang Q., Zhou F., Sun J, & Yang X. (2019). Establishment and Characterization of Two Novel Cholangiocarcinoma Cell Lines. Annals of Surgical Oncology, 26(12), 4134-4147.
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