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Bdfacsaria 3 platform

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The BDFACSAria III is a high-performance flow cytometry platform designed for cell sorting and analysis. It provides advanced capabilities for precise measurement and separation of cell populations based on their physical and fluorescent characteristics.

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Lab products found in correlation

Facsdiva software, by BD (4 mentions) Cd3e pe, by BD (1 mentions) Cd11c apc, by BD (1 mentions) Cd19 pe cy7, by BD (1 mentions) Direct zol rna extraction kit, by Zymo Research (1 mentions) Fcap array software v3, by BD (1 mentions)

7 protocols using bdfacsaria 3 platform

1

Quantification of Immune Cell Subsets

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Fresh isolated 2 × 106 cells/mouse of splenocytes or MLNLs for each group were analyzed by flow cytometry. Separated cells were dyed with rabbit anti-mouse CD16/CD32, CD3e-PE, CD19-PE-Cy7, and CD11c-APC (BD Biosciences, Shanghai, China), as well as goat anti-mouse CCR10-Axlexa Flour 488 (Abcam, Cambridge, UK), at 4 °C for 30 min. The stained cells were washed and detected on a BD FACSAria III platform. The absolute number of CD3e+, CD19+, or CD11c+ cells (1 × 105/mouse) from each immunization group and control group (pcDNA3.1) was analyzed by flow cytometry. CCR10 expression on CD3e+, CD19+, or CD11c+ cells was analyzed using FlowJo V10 software (BD Biosciences, Shanghai, China).
Yan Y., Hu K., Fu M., Deng X., Guan X., Luo S., Zhang M., Liu Y, & Hu Q. (2022). CCL28 Enhances HSV-2 gB-Specific Th1-Polarized Immune Responses against Lethal Vaginal Challenge in Mice. Vaccines, 10(8), 1291.
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2

CRISPR-Cas9 Mediated Genome Editing in Liver Cells

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The HepG2 and LO2 liver cells were purchased from American Type Culture Collection (ATCC) and maintained in DMEM supplemented with 10% foetal bovine serum. Transfection of 0.5 μg pX330 (Cas9‐sgCPS1) and 3 μg donor plasmid were made into 5 × 105 cells with lipofectamine 2000. Single cells were seeded into each well of a 96‐well plate using the BD FACSAria III platform and were subjected to selection conditions with 1 mg/ml G418 for 2 weeks.
Wang Y., Chang L., Zhai J., Wu Q., Wang D, & Wang Y. (2017). Generation of carbamoyl phosphate synthetase 1 reporter cell lines for the assessment of ammonia metabolism. Journal of Cellular and Molecular Medicine, 21(12), 3214-3223.
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3

FACS-Based Neuronal Nuclear Isolation

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Labelled nuclear extracts were processed the BDFACSAria III platform (BD Biosciences, San Jose, CA) according to technical specifications provided by the company. We used BD FACSDIVA software (BD Biosciences, San Jose, CA) to first isolate single, intact nuclei based on DRAQ5 fluorescence at the 730/45-A filter (DRAQ5), then to sort neuronal from non-neuronal nuclei based on fluorescence detected by the 585/42 filter (PE). Sorted nuclear fractions were stored at −20°C in sheath fluid (1X PBS) until DNA extraction. On average, we isolated 180 000 NeuN+ nuclei and 492 500 NeuN- nuclei from 50mg of tissue, with roughly 37% NeuN+ in each sample. There were no differences between cases and controls in terms of total nuclei in either fraction or in the ratio of neuronal to non-neuronal nuclei captured per dissection (ps > 0.1, Supplemental Figure 3b–d).
Vaillancourt K., Yang J., Chen G.G., Yerko V., Théroux J.F., Aouabed Z., Lopez A., Thibeault K.C., Calipari E.S., Labonté B., Mechawar N., Ernst C., Nagy C., Forne T., Nestler E.J., Mash D.C, & Turecki G. (2020). Cocaine-related DNA methylation in caudate neurons alters 3D chromatin structure of the IRXA gene cluster.. Molecular psychiatry, 26(7), 3134-3151.
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4

FACS-Based Neuronal Nuclear Isolation

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Labelled nuclear extracts were processed the BDFACSAria III platform (BD Biosciences, San Jose, CA) according to technical specifications provided by the company. We used BD FACSDIVA software (BD Biosciences, San Jose, CA) to first isolate single, intact nuclei based on DRAQ5 fluorescence at the 730/45-A filter (DRAQ5), then to sort neuronal from non-neuronal nuclei based on fluorescence detected by the 585/42 filter (PE). Sorted nuclear fractions were stored at −20°C in sheath fluid (1X PBS) until DNA extraction. On average, we isolated 180 000 NeuN+ nuclei and 492 500 NeuN- nuclei from 50mg of tissue, with roughly 37% NeuN+ in each sample. There were no differences between cases and controls in terms of total nuclei in either fraction or in the ratio of neuronal to non-neuronal nuclei captured per dissection (ps > 0.1, Supplemental Figure 3b–d).
Vaillancourt K., Yang J., Chen G.G., Yerko V., Théroux J.F., Aouabed Z., Lopez A., Thibeault K.C., Calipari E.S., Labonté B., Mechawar N., Ernst C., Nagy C., Forne T., Nestler E.J., Mash D.C, & Turecki G. (2020). Cocaine-related DNA methylation in caudate neurons alters 3D chromatin structure of the IRXA gene cluster.. Molecular psychiatry, 26(7), 3134-3151.
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5

Isolation and Characterization of Neuronal Nuclei

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Labeled nuclei were passed through 40 μM filter caps to remove any remaining cellular debris and processed using the BDFACSAria III platform (BD Biosciences) according to technical specifications provided by the company. We used BD FACSDIVA Software (BD Biosciences) to first isolate single, intact nuclei based on DRAQ5 fluorescence at the 730/45 A filter (DRAQ5), and then to sort neuronal from non-neuronal nuclei based on fluorescence detected by the 585/42 filter (PE). Sorted nuclear fractions were collected in sheath fluid (1×phosphate-buffered saline (PBS)). Upon sort completion, 3 X volume Qiazol was immediately added to each fraction and stored at –80 °C until RNA extraction. RNA was extracted using Direct-zol RNA extraction kit (Zymo) following manufacturer instructions. Due to larger volume of starting material a vacuum system was used instead of centrifugation for the initial RNA extraction step. cDNA conversion and SNORD90 quantification was same as detailed above with slight modifications. Instead of random hexamers, SNORD90 reverse primer (40 µM) was used in cDNA construction to aid in SNORD90 quantification via qPCR.
Lin R., Kos A., Lopez J.P., Dine J., Fiori L.M., Yang J., Ben-Efraim Y., Aouabed Z., Ibrahim P., Mitsuhashi H., Wong T.P., Ibrahim E.C., Belzung C., Blier P., Farzan F., Frey B.N., Lam R.W., Milev R., Muller D.J., Parikh S.V., Soares C., Uher R., Nagy C., Mechawar N., Foster J.A., Kennedy S.H., Chen A, & Turecki G. (2023). SNORD90 induces glutamatergic signaling following treatment with monoaminergic antidepressants. eLife, 12, e85316.
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6

Th1/Th2 Cytokine Response to gB Protein

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Splenocytes (1 × 107 per well, n = 5 mice per group) in 1 mL complete RPMI 1640 medium were added to 24-well plates. Purified gB protein (1 μg/mL) was added as a stimulant and cultured at 37 °C for 5 days. After incubation, filtered cell supernatants were used to detect the Th1/Th2 cytokines (IL-2, IL-4, IL-5, IFN-γ, and TNF-α) by mouse Th1/Th2 cytokine kits (BD Biosciences, Shanghai, China) according to the manufacturer’s instructions. The test and data acquisition were performed on the BD FACSAria III platform and analyzed by FCAP Array Software v3.0 (BD Biosciences, Shanghai, China).
Yan Y., Hu K., Fu M., Deng X., Guan X., Luo S., Zhang M., Liu Y, & Hu Q. (2022). CCL28 Enhances HSV-2 gB-Specific Th1-Polarized Immune Responses against Lethal Vaginal Challenge in Mice. Vaccines, 10(8), 1291.
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7

Neuronal Nuclei Isolation and Sorting

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Labeled nuclear extracts were processed on our in-house BDFACSAria III platform (BD Biosciences, San Jose, CA) according to technical specifications provided by the company. We used BD FACSDIVA software (BD Biosciences, San Jose, CA) to first isolate single, intact nuclei based on DRAQ5 fluorescence at the 730/45-A filter (DRAQ5), then to sort neuronal from non-neuronal nuclei based on fluorescence detected by the 585/42 filter (PE). Sorted nuclear fractions were stored at -20°C until DNA extraction.
Vaillancourt K., Chen G.G., Fiori L., Maussion G., Yerko V., Théroux J.F., Ernst C., Labonté B., Calipari E., Nestler E.J., Nagy C., Mechawar N., Mash D.C, & Turecki G. (2021). Methylation of the tyrosine hydroxylase gene is dysregulated by cocaine dependence in the human striatum. iScience, 24(10), 103169.
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