Facslyric flow cytometer

Manufactured by BD

Sourced in United States, Germany

The BD FACSLyric flow cytometer is a compact, automated instrument designed for the detection and analysis of cells or particles in liquid samples. It utilizes flow cytometry technology to measure multiple parameters of individual cells or particles as they pass through a laser beam. The core function of the BD FACSLyric is to provide accurate cell counting, sorting, and analysis capabilities for various applications in research and clinical laboratories.

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104 protocols using facslyric flow cytometer

NG2 Expression and Proliferation Assay

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The untreated JA cells were washed with phosphate-buffered saline (PBS) and harvested with an enzyme-free dissociation buffer (Thermo Fisher Scientific, Darmstadt, Germany). The cells were incubated with a phycoerythrin (PE)-labeled primary anti-NG2 antibody for 30 min at room temperature. Thereafter, the cells were washed with PBS and the mean fluorescence intensity (MFI) of 1000 cells was analyzed by a FACSLyric™ flow cytometer (BD, Heidelberg, Germany). The non-expressing NG2 cell line HEK293 was used as a negative control.
To determine cell proliferation, the JA cells were treated with CX-4945 (10 µM), SGC-CK2-1 (5 µM) or DMSO and exposed to 10 µM BrdU for 48 h. Thereafter, the cells were fixed, permeabilized and incubated with an anti-BrdU antibody. The cells were then washed with PBS and the MFI of 500 cells was analyzed by a FACSLyric™ flow cytometer (BD, Heidelberg, Germany).

Boewe A.S., Wemmert S., Kulas P., Schick B., Götz C., Wrublewsky S., Montenarh M., Menger M.D., Laschke M.W, & Ampofo E. (2022). Inhibition of CK2 Reduces NG2 Expression in Juvenile Angiofibroma. Biomedicines, 10(5), 966.

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Quantifying Apoptotic Glioma Cells

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The quantification of cell numbers was performed using the BD FACSLyric™ flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA), which utilizes its fixed-speed-aspiration capability to count cells. Initially, the supernatant containing both floating and dead cells was collected. To inactivate trypsin and prevent potential cell loss, the collected supernatant from each sample was used for cell resuspension. To avoid any loss of cells, the harvested cells were neither washed nor centrifuged. A portion of each sample was then vortexed and immediately measured for a duration of 45 s. The total number of cells in the sample was calculated based on the count obtained from the measured sample fraction, considering only events that corresponded to cells of normal size, with debris excluded.
Apoptotic glioma cells were detected using Annexin V staining. Cells were first trypsinized, then washed and resuspended in 50 µL of Annexin V binding buffer (pH 7.4) containing 10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl 2 . The cells were then incubated with 1.25 µL of APC-conjugated Annexin V probe (BD Biosciences, Cat. # 550475) for 15 min at room temperature. Following incubation, the cells were analyzed using the BD FACSLyric™ flow cytometer (BD Biosciences) to identify and quantify apoptotic cells.

Sharapova G., Sabirova S., Gomzikova M., Brichkina A., Barlev N.A., Kalacheva N.V., Rizvanov A., Markov N, & Simon H.U. (2024). Mitochondrial Protein Density, Biomass, and Bioenergetics as Predictors for the Efficacy of Glioma Treatments. International journal of molecular sciences, 25(13).

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Assessing Cellular Oxidative Stress Levels

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The cell-permeant 2’,7’-dichlorodihydrofluorescein diacetate (H₂DCFDA) fluorescent probe (Thermo Fisher Scientific) was used to evaluate the intracellular levels of reactive oxygen species. R1 (10⁵ cells) and fibroblast 3T3 (2×10⁵ cells) were seeded in 10 cm Petri dishes and exposed to 0.3 mM or 0.6 mM CYP, respectively, for 8 (3T3 only), 12 (R1 only), 24, 48 and 72 h (both cell lines). Then, cells were collected, centrifuged at 500 rpm for 5 min and resuspended in 1x PBS solution containing 10 μM H₂DCFDA. The mixture was incubated at 37oC for 30 min under dark conditions. The analyses of the samples were performed using a flow cytometer FACS Lyric BD (BD Biosciences). A minimum of 50,000 events were acquired for each sample. Data were processed, plotted and analysed using the BD FACSuite^TM flow cytometer software.

Rebuzzini P., Civello C., Akono E.N., Fassina L., Zuccotti M, & Garagna S. (2020). Chronic cypermethrin exposure alters mouse embryonic stem cell growth kinetics, induces Phase II detoxification response and affects pluripotency and differentiation gene expression. European Journal of Histochemistry : EJH, 64(1), 3084.

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Cell Cycle Analysis of R1 and 3T3 Cells

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R1 (10⁵ cells) and fibroblast 3T3 (2×10⁵ cells) were seeded in 10 cm Petri dishes and exposed to 0.3 mM and 0.6 mM CYP, respectively. After 8 (3T3), 12 (R1), 24, 48 and 72 h, the cells were harvested, washed in 1x PBS and stained with 1.5 ml of 50 μg/ml propidium iodide (PI), containing 100 U/ml of RNase and 0.05% Igepal (Sigma-Aldrich).
The analyses of the samples were performed using a flow cytometer FACS Lyric BD (BD Biosciences). A minimum of 50,000 events were acquired for each sample. Data were processed, plotted and analysed using the BD FACSuite^TM flow cytometer software.

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T Cell Activation Protocol with Inhibitors

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To induce T lymphocyte activation, 1 × 10⁶ cells were grown in RPMI-1640 5% FBS at 37 °C with 5% CO₂ in a humidified atmosphere supplemented with polyclonal activators 50 ng/mL phorbol 12-myristate 13-acetate (PMA) plus 1 μM Ionomycin A (Ion A) (Sigma Aldrich) for 2 h and 6 h. Activation of T cells receptor (TCR) was carried out with antibodies anti-CD3 and anti-CD28 bound to magnetic beads (Dynabeads™, Thermofisher). The percentage of activated cells was measured at 48 h by BD FACSLyric™ flow cytometer (Becton Dickinson) with BD Multitest™ CD8/CD38/CD3/HLA-DR and anti-CD4 PECy7 and anti-CD45 V500C (Becton Dickinson) (Additional file 1: Fig S1).
The mean percentage of activated cells was 33% (SD = ± 8,2%).
RPMI-1640 was supplemented with IL-2 (30U/mL)(Merck), IL-4 (200 ng/mL), IL-6 (200 ng/mL), IFN-α (180 ng/mL) and IFN-γ (200 ng/mL)(GIBCO™, Thermofisher) where described.
PI3K inhibitor, LY294002 (Ly) or Wortmannin (Calbiochem), were used at final concentration of 10 μM and 1 µM, respectively. The MEK1/2 inhibitor PD98059 (PD) and the Src kinase inhibitor PP1 (Alexis) were used at a final concentration of 5 µM.

Di Zazzo E., Rienzo M., Casamassimi A., De Rosa C., Medici N., Gazzerro P., Bifulco M, & Abbondanza C. (2023). Exploring the putative role of PRDM1 and PRDM2 transcripts as mediators of T lymphocyte activation. Journal of Translational Medicine, 21, 217.

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Multiparameter Bone Marrow Immunophenotyping in MM

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A total of 25 (out of 102) MM patients (n = 14 with low ferritin levels and n = 11 with high ferritin levels) presented BM available for further characterization and were enrolled for the flow cytometric immunophenotyping of bone marrow microenvironment using the BD OneFlow™ PCD and Plasma Cell Screening Tube (PCST) (Beckton Dickinson, Franklin Lakes, NJ, USA) containing the following fluorochrome-conjugated antibodies: anti-human CD38 FITC, anti-human CD28 PE, anti-human CD27 PerCP Cy5.5, anti-human CD19 PE-Cy7, anti-human CD117 APC, anti-human CD81 APC-H7, anti-human CD45 BD Horizon V450, anti-human CD138 BD Horizon V500-C, anti-human CD56 PE, anti-human b2-microglobulin PerCP Cy5.5, anti-human Kappa APC, and anti-human Lambda APC-H7 antibodies.
BM aspirates were collected in EDTA tubes and stained according to the producers’ guidelines. Once washed, samples were acquired on the BD FACSLyric™ Flow Cytometer (Beckton Dickinson, Franklin Lakes, NJ, USA).

Plano F., Gigliotta E., Corsale A.M., Azgomi M.S., Santonocito C., Ingrascì M., Di Carlo L., Augello A.E., Speciale M., Vullo C., Rotolo C., Camarda G.M., Caccamo N., Meraviglia S., Dieli F., Siragusa S, & Botta C. (2023). Ferritin Metabolism Reflects Multiple Myeloma Microenvironment and Predicts Patient Outcome. International Journal of Molecular Sciences, 24(10), 8852.

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Flow Cytometric Analysis of Proliferating Cells

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500 000 cells were fixed for 15 minutes at RT with 100 µL IntraStain reagent A (Dako-Agilent, CA, USA, cat: K2311). After washing (5 minutes, 400g, room temperature) with PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH = 7.4), the pellet was resuspended in 100 µL IntraStain reagent B containing 0.4% Triton X-100 and 5 µL Ki67 antibody (Sony Biotechnology, Weybridge, UK, Cat# 2352515, RRID:AB_2920575). After 15 minutes of incubation at room temperature in the dark, the cells were washed again (5 minutes, 400g, room temperature) and the pellet was resuspended in 0.5 mL PBS containing 0.01 mg/mL 2-(4-amidinophenyl)-6-indolecarbamidine (DAPI) and incubated for 30 minutes at room temperature in the dark. The samples were measured with a BD FACS Lyric flow cytometer (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). At least 50 000 events were acquired. Flow cytometry data were analyzed using Kaluza 2.1.1 software (Beckman Coulter, Brea, CA, United States).

Szabó B., Németh K., Mészáros K., Krokker L., Likó I., Saskői É., Németh K., Szabó P.T., Szücs N., Czirják S., Szalóki G., Patócs A, & Butz H. (2022). Aspirin Mediates Its Antitumoral Effect Through Inhibiting PTTG1 in Pituitary Adenoma. The Journal of Clinical Endocrinology and Metabolism, 107(11), 3066-3079.

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Flow Cytometric Analysis of Annexin V-FITC Apoptosis

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Analysis of phosphatidylserine externalization in apoptotic cells was determined using an Annexin V-FITC Apoptosis Detection kit (cat no. ab14085; Abcam, Cambridge, UK) according to the manufacturer’s protocol. Briefly, THP-1 cells were seeded in 6-well plates at a density of 1.0 × 10⁶ cell/well, and incubated with the different IC₅₀Rhus tripartita extracts at 37 °C or camptothecin 2 μM as positive control CTRL(+) at 5% CO₂ in a humid atmosphere for 48 h. At the end of the incubation, the cells were harvested, washed twice with PBS (1X) and stained with 5 μl of FITC Annexin V (Abcam, Cambridge, UK) and 5 μl of propidium iodide (PI; Abcam, Cambridge, UK) in the dark for 5 min at room temperature. Following this procedure, cell apoptosis was analyzed on BD FACS Lyric™ flow cytometer (Becton, Dickinson and Company, Franklin Lakes, New Jersey, USA) (Ex = 488 nm; Em = 530 nm) using FITC signal detector (FL1) and PI staining by the phycoerythrin emission signal detector (FL2). Data were analyzed with the BD FACSuite™ v1.2.1 software package (BD FACSuite™, Becton, Dickinson and Company, Franklin Lakes, New Jersey, USA).

Tlili H., Macovei A., Buonocore D., Lanzafame M., Najjaa H., Lombardi A., Pagano A., Dossena M., Verri M., Arfa A.B., Neffati M, & Doria E. (2021). The polyphenol/saponin-rich Rhus tripartita extract has an apoptotic effect on THP-1 cells through the PI3K/AKT/mTOR signaling pathway. BMC Complementary Medicine and Therapies, 21, 153.

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Apoptosis Detection in SH-SY5Y Cells

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The Annexin V-Cy3 Apoptosis Detection kit (Merck & Co., Inc.) was used to determine the number of apoptotic cells after treatment with HMW-αSo or LMW-αS. Differentiated SH-SY5Y cells exposed to 5 µM HMW-αSo or LMW-αS for 24 h were stained with Hoechst 33342 and Annexin V-Cy3, and then observed under a fluorescence microscope (BZ-X800). In addition, the cells were co-stained with Annexin V-FITC and PI (Thermo Fisher Scientific) and identified as early or late apoptotic and necrotic cells using a BD FACSLyric flow cytometer (Becton Dickinson, NJ, USA).

Ito N., Tsuji M., Adachi N., Nakamura S., Sarkar A.K., Ikenaka K., Aguirre C., Kimura A.M., Kiuchi Y., Mochizuki H., Teplow D.B, & Ono K. (2023). Extracellular high molecular weight α-synuclein oligomers induce cell death by disrupting the plasma membrane. NPJ Parkinson's Disease, 9, 139.

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Evaluating CD47 and PD-L1 Expression in CT-26 Cells

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To determine the effect of OXP and FOLFOX on cell surface expression of CD47 and PD-L1, CT-26 cell line (Pasture Institute, Tehran, Iran ) were grown in RPMI 1560 Gibco (NY, USA) with 10% FBS Gibco (NY, USA), penicillin and streptomycin then seeded to 6-well plate. After 24 h cell were treated either with 50 µM OXP (Sobhan, Tehran, Iran) or 50 µM OXP (Sobhan, Tehran, Iran) plus 10 µM 5-fluorouracil (FOLFOX regimen), (Alhavi, Tehran, Iran). The concentration of chemotherapeutic agent used for the in-vitro study was based on previous studies, which led to maximum ICD induction^{1 (link)}. Six hours after CT-26 tumor cells were treated with chemotherapeutic agents, cells were harvested, stained with fluorochrome-conjugated antibodies, and analyzed for cell surface expression of CD47 and PD-L1 with BD FACSlyric flow cytometer (Becton Dickinson, USA).

Alimohammadi R., Mahmoodi Chalbatani G., Alimohammadi M., Ghaffari-Nazari H., Rahimi A., Mortaz E., Mossafa N., Boon L, & Jalali S.A. (2023). Dual blockage of both PD-L1 and CD47 enhances the therapeutic effect of oxaliplatin and FOLFOX in CT-26 mice tumor model. Scientific Reports, 13, 2472.

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