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Fc500mcl

Manufactured by Beckman Coulter
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The FC500MCL is a flow cytometry system designed for research and clinical laboratory applications. It provides high-performance, multi-color analysis and sorting capabilities. The instrument is capable of detecting and measuring various cellular parameters, including size, granularity, and fluorescence intensity.

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Lab products found in correlation

Cxp analysis 2, by Beckman Coulter (3 mentions) Ab113851, by Abcam (2 mentions) Facscalibur, by BD (2 mentions) Fv1000, by Olympus (1 mentions) Facscalibur system, by Beckman Coulter (1 mentions) Hla dr, by BD (1 mentions) Trypsin, by Thermo Fisher Scientific (1 mentions) Annexin 5 fitc and pi, by BestBio (1 mentions) Dm4000b, by Leica (1 mentions) Lyticase, by Merck Group (1 mentions) Dihydroethidium dhe, by Cayman Chemical (1 mentions) Cytomics fc 500 mcl, by Beckman Coulter (1 mentions) Annexin 5 pi apoptosis kit, by Liankebio (1 mentions) Annexin 5 fitc apoptosis detection kit, by Abcam (1 mentions) Mitosox red, by Thermo Fisher Scientific (1 mentions) Anti cd24 fitc, by BD (1 mentions) Anti cd44 pe, by BD (1 mentions) Annexin 5, by MultiSciences Biotech (1 mentions) Annexin 5, by Beckman Coulter (1 mentions) Lsm 510 meta, by Zeiss (1 mentions)

47 protocols using fc500mcl

1

Quantifying ROS and Apoptosis in U87 Cells

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To detect ROS levels, 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA; Sigma-Aldrich; Merck KGaA) was diluted with serum-free DMEM (1:1,000; HyClone; Cytiva) to a final concentration of 10 µmol/l. U87 cells were collected by centrifugation at 400 x g, 4˚C for 5 min, suspended in diluted DCFH-DA (10 µmol/l) at 1x107 cells/ml and incubated at 37˚C for 20 min. The cell suspension was mixed every 3-5 min to fully integrate the probe with the cells. Following incubation, the cells were washed three times with serum-free medium to remove excessive DCFH-DA. The cells (1x106) were subsequently harvested by centrifugation at 400 x g, 4˚C for 5 min, and ROS levels were detected using flow cytometry (FC500 MCL; Beckman Coulter, Inc.). The data was analyzed using CXP analysis version 2.0 (Beckman Coulter, Inc.).
To detect apoptosis, U87 cells (1x106) were collected and washed with 1 ml pre-cooled PBS. Annexin V-FITC and propidium iodide (10 µl each; both Beijing Solarbio Science and Technology Co., Ltd.) were added to the cells and the percentage of apoptosis in the stained cells was quantified using flow cytometry (FC500 MCL; Beckman Coulter, Inc.). The data was analyzed using CXP analysis version 2.0 (Beckman Coulter, Inc.).
Xu Z., Zeng X., Li M., Liao J, & Chen Q. (2021). MicroRNA-383 promotes reactive oxygen species-induced autophagy via downregulating peroxiredoxin 3 in human glioma U87 cells. Experimental and Therapeutic Medicine, 21(5), 439.
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2

Evaluating ICAM-1 Expression and Targeting

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The EAs were stimulated with 400 ng/mL of LPS for 24 h to mimic the ECs under inflammatory pathological state. To qualitatively analyze the ICAM-1 expression via immunofluorescent staining, both of the quiescent EAs and activated EAs (LPS-stimulation for 24 h) were incubated with primary antibody (mouse anti-human ICAM-1 antibody) overnight at 4 °C. Afterwards, the cells were incubated with fluorescent secondary antibody (Alexa Fluor® 405-conjugated Goat Anti-Mouse IgG) for 60 min at room temperature. The nuclei were stained with PI. The cells were observed via a confocal microscopy (OLYMPUS FV1000) to investigate ICAM-1 expression on EAs surface after LPS stimulation.
To evaluate ICAM-1 targeting characteristic of anti-ICAM/SV/NLCs in vitro, the activated EAs were incubated with anti-ICAM/SV/NLCs and control IgG/SV/NLCs (20 μg/mL) simultaneously for 1 h. Meanwhile, EAs were pre-incubated with free ICAM-1 antibody (3 μγ/mL) for 1 h to block ICAM-1 epitope on the cells and then incubated with anti-ICAM/SV/NLCs. ODA-FITC was still employed as the fluorescent probe encapsulated in the formulated NLCs (ODA-FITC:lipid = 5:100, w/w). Afterwards, the EAs were harvested followed by washing with PBS via centrifugation. A flow cytometer (FC500MCL, Beckman Coulter) was performed to quantitatively analyze the uptake of formulated NLCs.
Li S.J., Wang X.J., Hu J.B., Kang X.Q., Chen L., Xu X.L., Ying X.Y., Jiang S.P, & Du Y.Z. (2017). Targeting delivery of simvastatin using ICAM-1 antibody-conjugated nanostructured lipid carriers for acute lung injury therapy. Drug Delivery, 24(1), 402-413.
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3

Phenotypic Characterization of Cultured MenSCs

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Surface markers of cultured MenSCs were analyzed using a flow cytometer (FC500MCL, Beckman Coulter, United States). The third and fifth passages of MenSCs ( 5 x 105 cells/100 μL) were incubated directly with phycoerythrin (PE) or allophycocyanin (APC)-conjugated mouse monoclonal antibodies against human CD9, CD29, CD41a, CD44, CD59, CD 73, CD90, CD105, CD14, CD34, CD45, CD117, and human leukocyte antigen (HLA)-DR (BD Biosciences, CA, United States) for 30 min in the dark at 4 °C, followed by washing and resuspension in PBS twice. Flow cytometry was conducted using a FACSCalibur system (FC500, Beckman Coulter, United States). Data were analyzed using FlowJo Version 10.05.
Cen P.P., Fan L.X., Wang J., Chen J.J, & Li L.J. (2019). Therapeutic potential of menstrual blood stem cells in treating acute liver failure. World Journal of Gastroenterology, 25(41), 6190-6204.
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4

Measuring Intracellular ROS and Mitochondrial Potential

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The levels of intracellular ROS and mitochondrial membrane potential were examined by flow cytometry, using H2DCFDA and JC-1, respectively. The procedures were described in a previous report [39 (link)]. Cells treated with CYT-Rx20 for 2 h were loaded with 20 μM H2DCFDA and those treated for 24 h were loaded with 2 μM JC-1, followed by incubation at 37°C for 30 min or 15 min (JC-1) in a dark environment. The cells were then analyzed immediately by flow cytometry (FC 500 MCL, Beckman Coulter, Brea, CA, USA).
Tsai C.H., Hung A.C., Chen Y.Y., Chiu Y.W., Hsieh P.W., Lee Y.C., Su Y.H., Chang P.C., Stephen Chu-Sung H, & Yuan S.S. (2017). 3’-hydroxy-4’-methoxy-β-methyl-β-nitrostyrene inhibits tumorigenesis in colorectal cancer cells through ROS-mediated DNA damage and mitochondrial dysfunction. Oncotarget, 8(11), 18106-18117.
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5

Quantifying Cellular Reactive Oxygen Species

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Total cellular ROS generation was detected with 20 µM 2′, 7′-dichlorodihydrofluorescein diacetate (ab113851, Abcam) using an FC500 MCL (Beckman Coulter), with excitation and emission wavelengths of 488 nm and 530 nm, respectively. According to the manufacturer’s instructions, 5 µM MitoSOX was added to the plates, followed by incubation for 10 min at 37 °C. The cells were then washed twice with pre-warmed (37 °C) HBSS Ca/Mg and detached with 0.25% trypsin. Fluorescence was measured using a FACSCalibur (BD Biosciences), with excitation and emission wavelengths of 488 nm and 580 nm. The data were analyzed with FlowJo software (FlowJo, LLC), version 10.
Emamdoust F., Aminafshar M., Zandi M, & Sanjabi M.R. (2022). The role of Rho-associated kinase inhibitor, Y-27632 on primary culture of ovine spermatogonial stem cells. Animal Reproduction, 18(4), e20200257.
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6

Intracellular Drug Uptake in Caco-2/HT-29/Raji-B Model

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Caco-2 cells at the logarithmic growth stage were inoculated in cell plates at a density of 2 × 105 cells/mL. The Caco-2/HT-29 cell model was established by inoculating Caco-2 and HT-29 cells at a population ratio of 7:3. The medium was changed every other day for the first week and daily from the second week. A total of 105 cells/well of Raji-B cells were added to establish Caco-2/HT-29/Raji-B three-cell model. After 14 days of incubation at 37 °C with 5% CO2, the medium was removed and the culture medium containing Rhodamine B, Rhodamine-CS-TPP (Rho-CS), or Rho-CS-TPP-ALG NPs (Rho-NPs) was added with a final drug fluorescence concentration of 1 μg/mL. After incubation for 4 h, the drug-containing culture medium was discarded and the cells were washed with PBS three times. Subsequently, trypsin containing 0.2% EDTA was added to digest the cells, which were collected, centrifuged, and re-suspended with 0.5 mL of PBS. The intracellular drug uptake was determined by flow cytometry (FC500MCL, Beckman, MA, USA).
Yang J.M., Wu L.J., Lin M.T., Lu Y.Y., Wang T.T., Han M., Zhang B, & Xu D.H. (2022). Construction and Evaluation of Chitosan-Based Nanoparticles for Oral Administration of Exenatide in Type 2 Diabetic Rats. Polymers, 14(11), 2181.
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7

Annexin V-FITC Apoptosis Assay

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Apoptosis degree was measured by Annexin V staining (Biosharp Biotechnology (Shanghai, China)). Firstly, A549 cells were incubated with the tested compound (arenobufagin) for 48 h, then collected from six-well plates, washed once with cold PBS (4C), and centrifuged (2000 rpm for 5 min) before being suspended with a 300 μL binding buffer. Annexin V-FITC (6 μL) was then added and the cells were stained in the dark for 15 min before adding 6 μL propidium iodide and 300 μL binding buffer. Apoptosis quantification was counted by flow cytometry (FC 500MCL, Beckman Coulter, Indianapolis, IN, USA).
Wu J.H., Cao Y.T., Pan H.Y, & Wang L.H. (2020). Identification of Antitumor Constituents in Toad Venom by Spectrum-Effect Relationship Analysis and Investigation on Its Pharmacologic Mechanism. Molecules, 25(18), 4269.
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8

Mitochondrial Degradation and Cell Viability

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Cell growth of treated and untreated cells was followed by measuring culture OD660. Cell viability with treatment for 3 h was assayed by counting colony-forming units (CFU) on YPD agar plates after 2 d at 30 °C. Mitochondrial degradation was measured by quantifying mitochondrial GFP fluorescence using a FC500MCL flow cytometer (Beckman Coulter, USA)11 (link).
Dong Y., Hu J., Fan L, & Chen Q. (2017). RNA-Seq-based transcriptomic and metabolomic analysis reveal stress responses and programmed cell death induced by acetic acid in Saccharomyces cerevisiae. Scientific Reports, 7, 42659.
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9

Cellular Uptake of FITC-Insulin Formulations

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For quantitative study, MDCK cells were seeded at a density of 1 × 105 cells per well into a 12-well culture plate and allowed to attach for 24 h until confluence. The medium was then removed and treated with fresh MEM containing free FITC-INS or FITC-INS-PEOP (10 μg mL−1) at 37 °C for 1 h and 4 h. After incubation, the cells were washed with PBS (pH 7.4), detached with trypsin–EDTA and resuspended in proper volume of PBS for flow cytometer analysis (Beckman Coulter, FC500MCL, US). Data from 10 000 events were gated using forward and side scatter parameters to exclude debris and dead cells as well as control cells incubated with media alone as control for autofluorescence. All experiments were conducted in triplicate and data presented as means ± standard deviation.
Hu Y., Wang J, & Qiu L. (2020). Polymeric nano-vesicles via intermolecular action to load and orally deliver insulin with enhanced hypoglycemic effect. RSC Advances, 10(13), 7887-7897.
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10

Apoptosis Assay in Transfected Cells

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Transfected cells were collected and digested with 0.25% trypsin (Gibco). After digestion, they were washed twice with PBS and added with 100 μL binding buffer. They were configured into 1*106/mL suspension, and Annexin V-FITC and PI (BestBio, Shanghai, China) were successively added. The cells were incubated at room temperature in the dark for 5 min. The FC500MCL flow cytometry system (Beckman Coulter, USA) was used for determination, and the experiment was repeated three times to take the average value.
Wei M.C., Wang Y.M, & Wang D.W. (2021). miR-130a-Mediated KLF3 Can Inhibit the Growth of Lung Cancer Cells. Cancer Management and Research, 13, 2995-3004.
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