WT and ΔfepA P cells harboring various reporter plasmid constructs, were grown till 0.5 OD600 in LB at 37°C, then collected by centrifugation, and resuspended in PBS. Cells were sorted on a BD Aria Fusion flow cytometer with BD FACSDiva (v.9.0.1) software (BD Biosciences) using a 488 nm excitation laser (530/30 band pass) and a gating strategy as illustrated in Extended Data Fig. 5a (see also Fig. 4c ). The FACS sorter cannot deposit mother cells directly on a plate, so each sorted cell was deposited in one well of a 96-well plate prefilled with 20 μl LB, and then used for follow-up experiments as illustrated in Fig. 4d .
Aria fusion flow cytometer
Manufactured by BD
Sourced in United States
The BD Aria Fusion is a flow cytometer designed for high-performance cell analysis and sorting. It provides accurate and reliable data on multiple cellular parameters simultaneously. The core function of the BD Aria Fusion is to facilitate the analysis and separation of complex cell populations.
Automatically generated - may contain errors
Lab products found in correlation
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Lsm 780 confocal microscope, by Zeiss (3 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Facsdiva, by BD (1 mentions)
Complete edta free, by Roche (1 mentions)
Aria fusion, by BD (1 mentions)
Ficoll hypaque gradient, by GE Healthcare (1 mentions)
Streptavidin apc, by BioLegend (1 mentions)
Cd19 microbeads kit, by Miltenyi Biotec (1 mentions)
Streptavidin fitc, by BioLegend (1 mentions)
Puromycin, by BD (1 mentions)
Puromycin, by Merck Group (1 mentions)
Rosettesep, by STEMCELL (1 mentions)
Live dead fixable near ir dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Cellquest pro software, by BD (1 mentions)
Fam flica caspase 3 7 kit, by Bio-Rad (1 mentions)
10 protocols using aria fusion flow cytometer
1
Single-Cell Sorting and Culturing
2
Fluorescence-Activated Nuclear Sorting
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Cell-type specific nuclear purification was performed using fluorescent activated nuclear sorting56 (link),57 (link). Briefly, frozen striatal tissue was homogenized in ice-cold PBS supplemented with 1× Protease Inhibitors Cocktail (PIC, cOmplete EDTA free, Roche) and cross-linked in 1% formaldehyde for 15 min at room temperature. Cross-linking was stopped by the addition of glycine to final concentration 0.125 M and tissue was washed using ice-cold PBS. Cells were then lysed in Cell Lysis Buffer (10 mM Hepes pH 8; 85 mM KCl; 0.5% NP-40) and nuclei were collected after treatment with Nuclear Extraction Buffer (0.5% SDS, 10 mM EDTA pH 8, 50 mM Tris). Purified nuclei were then resuspended in PBTB (PBS 1×, 5% BSA, 0.5% Tween-20) + 1× PIC, 3% Normal Horse Serum (NHS) and stained using antibody to NeuN (1:1000, Merck Millipore). After washing, nuclei were labelled with Alexa Fluor 488 donkey anti-mouse IgG antibody(1:1500) and washed with ice-cold PBS. Immunostained nuclei were sorted using BD Aria Fusion flow cytometer, recovered in ice-cold 1× PBS, pelleted and stored at −80 °C for posterior ChIP-seq experiments.
3
Isolation and Characterization of SARS-CoV-2-Specific B Cells
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Peripheral blood mononuclear cells (PBMCs) were isolated immediately from fresh blood by Ficoll-Hypaque gradient (GE Healthcare) centrifugation. CD19+ B cells were enriched from pooled PBMCs using a CD19 MicroBeads kit (Miltenyi). The enriched CD19+ B cells were then stained with PE anti-human CD27 antibody (BD Biosciences, Cat# 566944, Clone name: O323, 1:50 dilution), SARS-CoV-2 biotinylated RBD protein (His-tagged) conjugated with FITC-streptavidin (Biolegend, Cat# 405202, 1:20 dilution), and biotinylated S1 protein (His-tagged) conjugated with APC-streptavidin (Biolegend, Cat# 405243, 1:20 dilution). CD19+CD27+RBD+S1+ B cells were sorted with a BD AriaFusion flow cytometer. The purity of sorted cells was rechecked by FACS again on a BD AriaFusion. Sorted CD27+Delta-S1+Delta-RBD+ B cells were resuspended in PBS containing 2% (v/v) fetal bovine serum (FBS) for future use. Flow cytometry data were analyzed using FlowJo v10.
4
CRISPR-Mediated B7H6 Knockout in Pancreatic Cancer
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To knock out B7H6 in pancreatic tumor cells, we performed CRISPR/Cas9 genome editing, using the all-in-one plasmid (06182015MN, Sigma-Aldrich). Briefly, Capan-1 and PANC-1 cells were transfected with the CRISPR plasmids, using Lipofectamine 3000 (L3000001, Thermo Fisher). Puromycin (10 μg/mL) (P8833-25MG Sigma-Aldrich) was used subsequently to select the edited cells. Puromycin-resistant cells were then FACS-sorted into singlets, using a BD Aria Fusion flow cytometer. The edited cells were then expanded and verified by immunoblotting analysis.
5
Regulatory T-cell Methylation Analysis
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Three SLE patients from cohort 2 (enrolled from CBR) were recalled based on the presence of a type 1 IFN signature and high frequency of CD25low FOXP3+ T cells. Total CD4+ T cells were enriched from 50 ml whole blood using RosetteSep (StemCell Technologies) and immunostained with fluorochrome-conjugated antibodies as described above (see Supplementary Table 1 ). Memory CD45RA− FOXP3+HELIOS+ Tregs from the (i) CD127lowCD25low and (ii) CD127lowCD25high subpopulations and a negative control subset of CD45RA− CD127+CD25int/low Teffs were FACS sorted using a BD Aria Fusion flow cytometer (BD Biosciences). Methylation of the FOXP3 TSDR was performed using a next-generation sequencing method, as described previously (26 (link)).
6
Cmab-h(m)CCL5 regulation of macrophage cytokines
7
Isolation and Culture of CD34+ Cells
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Thawed PBMCs were resuspended to 1 × 108 cells/mL in PBS supplemented with 2% FBS. Cells were stained with CD34 antibodies (Table 1 ) and LIVE/DEAD™ Fixable Near-IR Dead Cell Stain Kit (Invitrogen) in the dark on ice for 30 min to enable sorting of viable CD34+ cells. Stained cells were washed with PBS and resuspended at a final concentration of 1 × 107 cells/ml in 2% FBS. Cell sorting was performed with an Aria Fusion flow cytometer (BD Biosciences) at 4 C. Purified CD34+ viable cells were collected in 10% FBS in RPMI prior to seeding in drug-loaded 384-well plates and incubated for 20 h at 37 °C and 5% CO2. Finally, cells were fixed, blocked, and stained as described above in “MF PBMC immunofluorescence”.
8
Visualizing Nanoparticle-Mediated Optoporation
9
Macrophage Cytokine Gene Expression
10
Caspase 3/7 Activity Assay in Chlamydia-Infected Cells
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Caspase 3/7 activity assays were performed using the FAM FLICA™ Caspase-3/7 Kit (Bio-Rad) combined with propidium iodide (PI). McCoy cells were divided into 4 experimental groups. Half of the McCoy cells were infected with 0.01 MOI C. pneumoniae, while the other half were left uninfected. Both the infected and uninfected groups were subdivided into those treated with 0.05-mg/mL Ax or left untreated. After 12 h of incubation, the apoptosis assay was performed according to the manufacturer’s instructions, and the cells were analyzed via flow cytometry. The assay discriminated between viable (caspase 3/7−/PI−), early apoptotic (caspase 3/7+/PI−), and late apoptotic (caspase 3/7+/PI+) cells. The fluorescence of the cell populations was analyzed immediately using a BD fluorescence-activated cell sorting Aria Fusion flow cytometer (BD biosciences).
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