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Facscan

Manufactured by Thermo Fisher Scientific
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The FACScan is a flow cytometer designed for cell analysis. It utilizes laser technology to detect and analyze the physical and fluorescent characteristics of cells or particles suspended in a fluid stream.

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Lab products found in correlation

A11013, by Thermo Fisher Scientific (1 mentions) A32723, by Thermo Fisher Scientific (1 mentions) Live cell imaging solution, by Thermo Fisher Scientific (1 mentions)

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FACScan flow cytometer (4 citations)

6 protocols using facscan

1

Analyzing ALX Expression in Myofibroblasts

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Fibroblasts from each culture were seeded in 75 cm2 flasks at an initial density of 106 cells per flask. When the cells became 80% confluent, they were starved overnight and differentiated into myofibroblasts as described above. The medium was aspirated and the monolayer was washed twice with PBS. Cells were detached using trypsin/EDTA; spun at 500×g for 10 minutes, washed with PBS, and fixed with 4% paraformaldehyde. Two flasks were used for each experimental condition. Each flask yielded approximately 2×106 cells. In order to determine the expression of ALX (a.k.a. FPR2), cells (4×105) were treated with antibody buffer (5% normal donkey serum in PBS containing 0.03% sodium azide) and stained with phycoerythrin (PE)-conjugated mouse anti-human ALX/FPR2 antibody (1:200, Santa Cruz Biotechnology, Dallas, TX, USA) or isotype control (1:200), which is known to cross-react with mouse ALX. The cells were analyzed by fluorescence with FACScan (CellQuest software, eBiosciences, San Diego, CA, USA).
Herrera B.S., Kantarci A., Zarrough A., Hasturk H., Leung K.P, & Van Dyke T.E. (2015). LXA4 Actions Direct Fibroblast Function and Wound Closure. Biochemical and biophysical research communications, 464(4), 1072-1077.
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2

T Cell Phenotypic Analysis

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A phenotypic analysis of T cells was performed on a FACScan, as previously described.12 (link) T cells were harvested and stained with CYChrome-conjugated anti-mouse CD4 and PE-conjugated anti-mouse CD25 monoclonal antibody (eBioscience, San Diego, CA, USA).
Suzuki M., Yokota M., Nakamura Y., Ozaki S, & Murakami S. (2016). Intranasal administration of IL-35 inhibits allergic responses and symptoms in mice with allergic rhinitis. Allergology International, 66(2), 351-356.
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3

Siglec-15 Expression Analysis

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Hek293-hsiglec-15 or U87 cells were harvested and incubated with anti-Siglec-15 mAbs (from 0 to 10,000 ng/mL or 0 to 45,000 ng/mL) for 1 hour at 4°C. After incubation, the samples were washed with PBS 3 times and stained with Alexa488-conjugated anti-human (Invitrogen #A-11013) or mouse (Invitrogen #A32723) secondary antibodies in the dark for 50 minutes at 4°C. The mean fluorescence intensity of cells was measured by FACScan (Thermo). The data were analyzed using FlowJo software.
Ding H., Yao B., Ci L., Feng J., Ouyang P., Chen G., Hui X, & Zhou D. (2023). Enhancing the Anti-tumor Potency of a Novel Siglec-15 Antibody by Engineering its Fc-mediated Effector Functions. Journal of Immunotherapy (Hagerstown, Md. : 1997), 46(5), 161-169.
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4

Receptor Internalization Quantification by FACS

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Receptor internalization was quantitatively assessed using FACS. Hek293-hsiglec-15 cells were collected and seeded at 3–6×105 cells per well in a 6 well plate. Antibodies (10 μg/mL) were incubated with cells for 1 hour on ice to enable specific binding to the cell surface targets. After incubation, the cells were washed 3 times with cold PBS to remove unbound antibodies. The cells were then incubated at 37°C for 20 hours with or without antibodies to allow endocytosis to occur. Alexa488-conjugated secondary antibody was used to detect the binding of antibodies to the cells. The mean fluorescence intensity of cells was measured by FACScan (Thermo). FlowJo software was used to analyze the flow cytometry data.
Ding H., Yao B., Ci L., Feng J., Ouyang P., Chen G., Hui X, & Zhou D. (2023). Enhancing the Anti-tumor Potency of a Novel Siglec-15 Antibody by Engineering its Fc-mediated Effector Functions. Journal of Immunotherapy (Hagerstown, Md. : 1997), 46(5), 161-169.
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5

Assessing Ferroptosis with Erastin and Ubiquinol

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B4G12 cells were seeded at 100,000 cells/well in a 6-well plate in endothelial cell growth media supplemented with 10 μg/L of bFGF. After incubation at 37° C and 5% CO2 for 18 h, medium was removed from wells and ubiquinol, ubiquinol/γ-CD complex, or γ-CD, all in culture media, were added at 1, 10, 50, and 100 μM concentrations (n = 3). After incubation for 24 h at 37° C and 5% CO2, 10 μM of erastin in DMSO was added to all wells after washing cells twice with 1X DPBS, except untreated and untreated-unstained groups, and the plates were incubated at 37° C and 5% CO2 for 24 h. After incubation, 2 μl of C11-Bodipy 581/591 stock (in DMSO) were added to each well (except the untreated-unstained group), mixed vertically, and incubated for 20 min at 37° C and 5% CO2. Cells were then washed in 1X DPBS, detached by trypsin, transferred to 50 ml Falcon tubes, and washed again in 1X DPBS by centrifugation at 230 xg for 5 min. Finally, cells were resuspended in Live Cell Imaging Solution (Thermo Fisher), and the fluorescence was analyzed using flow cytometry (FACScan). Data analyzed was performed using FlowJo.
Naguib Y.W., Saha S., Skeie J.M., Acri T., Ebeid K., Abdel-rahman S., Kesh S., Schmidt G.A., Nishimura D.Y., Banas J.A., Zhu M., Greiner M.A, & Salem A.K. (2021). Solubilized Ubiquinol for Preserving Corneal Function. Biomaterials, 275, 120842.
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6

Siglec-15 Binding Assay

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Briefly, the hsiglec-15-Fc protein was incubated with anti-Siglec-15 mAbs or isotype mIgG (Biointron #B115101) on ice for 30 minutes, respectively, followed by incubation with Hek293-LRRC4C cells (2×105 cells/sample) on ice for 50 minutes. Cells were then washed with PBS to remove the unbound hsiglec-15-Fc. After washing, samples were stained with Alexa488-conjugated anti-human secondary antibodies (Invitrogen #A-11013) in the dark for 40 minutes at 4°C. The fluorescently labeled cells were measured by FACScan (Thermo). The data were analyzed using FlowJo software.
Ding H., Yao B., Ci L., Feng J., Ouyang P., Chen G., Hui X, & Zhou D. (2023). Enhancing the Anti-tumor Potency of a Novel Siglec-15 Antibody by Engineering its Fc-mediated Effector Functions. Journal of Immunotherapy (Hagerstown, Md. : 1997), 46(5), 161-169.
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