For detection of ploidy populations, primary hepatocytes (2 × 106/mL) were fixed in 75% ethanol at −20°C, then incubated with 500 μL (2 × 106 mL) of propidium iodide (PI)/RNase Staining Buffer (BD Pharmingen, BD 550825) at 25°C for 15 minutes. For RNA extraction from different ploidy populations, hepatocytes were fixed in 4% paraformaldehyde for 15 minutes, then permeabilized with 0.5% Triton X-100 for 30 minutes and stained with 4’,6-diamidino-2-phenylindole (DAPI). Cells were analyzed or sorted with a FACS Aria Fusion machine (BD Biosciences, San Jose, CA).
Facs aria fusion machine
Manufactured by BD
The BD FACS Aria Fusion is a high-performance cell sorting instrument designed for advanced flow cytometry applications. It features multicolor detection capabilities, allowing users to analyze and sort a wide range of cell types and populations. The core function of the BD FACS Aria Fusion is to provide reliable and efficient cell sorting for research and clinical laboratories.
Automatically generated - may contain errors
Lab products found in correlation
Pi rnase staining buffer, by BD (2 mentions)
Pre sort buffer, by BD (1 mentions)
Facsaria fusion, by BD (1 mentions)
Accutase, by Merck Group (1 mentions)
Colorless go taq buffer, by Promega (1 mentions)
Pe conjugated annexin 5, by BioLegend (1 mentions)
Hoechst, by Thermo Fisher Scientific (1 mentions)
Anti cd11b, by BD (1 mentions)
Anti ly 6g 6 c, by BD (1 mentions)
Anti cd3e, by BD (1 mentions)
Anti cd41, by BD (1 mentions)
Anti cd45r, by BD (1 mentions)
Trueprep dna library prep kit v2, by Illumina (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Novaseq 6000 instrument, by Illumina (1 mentions)
Ampure xp bead, by Beckman Coulter (1 mentions)
7 protocols using facs aria fusion machine
1
Hepatocyte Ploidy Analysis Protocol
2
Sorting and Characterization of Neuronal Cell Populations
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Fibroblast cells were detached by Accutase (Millipore) and resuspended in Pre‐Sort buffer (BD Biosciences). Red Fluorescent Protein (RFP) positive cells were aseptically sorted in a FACS ARIA Fusion machine (BD Biosciences) using a 130 μm nozzle at 20 psi. Cells were sorted in a 6‐well plate in prewarmed fibroblast growth medium, 2,000 RFP positive cells per well. Determination of RFP negative and RFP positive populations, after doublet discrimination, was based on gating of unelectroporated and electroporated cells in 616/23 and 695/40 filters.
Estimation of populations of forebrain and midbrain cells was done by flow cytometry on BD FACS Aria Fusion. Mature neurons were prepared for FACS as described, stained with DAPI and labeled with a TUJ1 antibody coupled to Alexa488 (TUJ1‐Alexa488). Forebrain cells we also labeled with MAP2‐Alexa647, and midbrain cells were labeled with TH‐Alexa647. Population of single cells was identified by doublet discrimination followed by DAPI positive gating. Neuronal and non‐neuronal populations were identified by gating of TUJ1‐Alexa 488 positive and negative populations of cells, respectively. MAP2+ and TH+ populations were identified by gating from TUJ1+ population.
Estimation of populations of forebrain and midbrain cells was done by flow cytometry on BD FACS Aria Fusion. Mature neurons were prepared for FACS as described, stained with DAPI and labeled with a TUJ1 antibody coupled to Alexa488 (TUJ1‐Alexa488). Forebrain cells we also labeled with MAP2‐Alexa647, and midbrain cells were labeled with TH‐Alexa647. Population of single cells was identified by doublet discrimination followed by DAPI positive gating. Neuronal and non‐neuronal populations were identified by gating of TUJ1‐Alexa 488 positive and negative populations of cells, respectively. MAP2+ and TH+ populations were identified by gating from TUJ1+ population.
3
Isolation and Amplification of B Cell Receptor Rearrangements
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Bone marrow B cells from three WT or CGI-Eμ mice were stained with anti-B220 APC, anti-CD43 PE and anti-IgM FITC antibodies and individual B220+IgM−CD43high cells were FACS sorted using a BD FACS Aria Fusion machine directly into 96-well PCR plates containing 1× Colorless Go Taq buffer (Promega), 0.5 mg/ml proteinase K, 10 μg/ml tRNA and water. The plates were then incubated at 55°C for 1 h, at 95°C for 10 min, and subjected to qPCR using Sso Fast Eva Green. Each plate was used to amplify a single D–JH rearrangement.
4
Isolation of Transgenic olig2+ Cells
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The transgenic line Tg (olig2:dsRed) was outcrossed to WT fish, and embryos were anesthetized at 28, 42, and 60 hpf with 0.04% MS-222 and dechorionated. Heads were removed rostral to the hindbrain-spinal cord boundary using a 24 G needle. The remaining trunks were deyolked in deyolking buffer (55 mM NaCl, 1.8 mM KCl, and 1.25 mM NaHCO3) and minced with fine scissors. Samples were dissociated with 0.25% trypsin at 28 °C for 30 min followed with 1 mg/ml collagenase II for 20 min. 10% fetal bovine serum was added to terminate the enzymatic reaction. Samples were passed through a 70 μm strainer and sorted with a BD FACS-Aria fusion machine as previously reported (51 (link)). FACS was performed in the core facility of Shanghai Institute of Nutrition of Heath, Chinese Academy of Sciences. FACS gating scatter plots with gating strategy was provided in the Fig. S9 .
5
Ploidy Analysis of Transfected Cells
6
Isolation of Fetal Liver Cell Populations
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To isolate cells of different stages of differentiation directly from primary fetal livers at E12.5–E13.5, 5–15 fetal livers were pooled together, mechanically dissociated in staining buffer (PBS, 0.2% BSA, 5 mM glucose) and strained through a 30-μM filter. Cells were stained at 4 °C with rabbit IgG (200 μg/ml, Jackson Laboratories) to block Fc receptors. Cells were then incubated with PE-Cy7 conjugated anti-CD71 (RI7217 BioLegend, 1:2000), APC conjugated anti-Ter119 (RUO, BD Biosciences; 1:200), BB700 conjugated CD117 (104D2, BD Biosciences, 1:100), PE-conjugated Annexin V (BioLegend, 1:100), a panel of 5 FITC-conjugated lineage antibodies (anti-CD41, anti-CD45R, anti-CD3e, anti-CD11b, and anti-Ly-6G/6C, all at 1 μg/ml, all BD Biosciences), Cells were then resuspended in FACS running buffer (staining buffer with the addition of 2 mM EDTA). 0.66 μg/ml Hoechst (Invitrogen) was added prior to sorting, which was performed on a BD FACSAria Fusion machine with a 100 μm nozzle. Annexin V was only added for the experiments on the Lrf−/− mice. Single color controls and FMOs were used for calibration.
7
Transcriptomic Analysis of Hamster Lung AMs
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Hamster lungs were collected 2 months post-vaccination and sorted for bronchoalveolar lavage AMs using a BD FACS Aria Fusion machine. One hundred thousand sorted cells were centrifuged at 500 × g for 10 min at 4 °C per replicate. Cells were lysed with lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM MgCl2, and 0.1% IGEPAL CA-630). Libraries were prepared using the TruePrep DNA library prep kit V2 for Illumina (Vazyme) according to the manufacturer’s instructions. Libraries were cleaned up with AMPure XP beads (Beckman coulter) at a ratio of 0.7 and the quality was assessed using the 2100 Bioanalyzer (Agilent Technologies). Libraries were sequenced with 150 paired ends using a NovaSeq 6000 instrument (Illumina) for an average of 20 million reads per sample.
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