mApple-LAMP1-pHluorin-N-8 was a gift from Dr. Michael Davidson (Addgene #54918). A LifeAct expressing plasmid was a gift from Dr. Janis Burkhardt (University of Pennsylvania). The LeGO-E plasmid containing Emerald-GFP was a gift from Dr. Boris Fehse (Addgene #27359) and LifeAct was cloned into BamHI and EcoRI restriction sites to create LifeAct.mEmerald. mTurquoise was a gift from Dr. Theodorus Gadella (University of Amsterdam). LifeAct.mTurquoise was generated by cloning LifeAct into the XhoI and EcoRI restriction sites of MIGR1 mTurquoise. NK92 cell lines were generated by retroviral transduction as previously described [16 (link)] or nucleofection using Amaxa Kit R per manufacturer’s instructions (Lonza). Positive cells were amplified under antibiotic selection pressure and were sorted for low, intermediate or high expression of the fluorescently tagged protein on an Aria II Fluorescence Activated Cell Sorter (BD). Each sorted population was then used for pilot experiments to determine the lowest possible expression level required for optimal imaging conditions by confocal, STED, TIRF or SIM.
Aria 2 fluorescence activated cell sorter
Manufactured by BD
Sourced in United States
The Aria II fluorescence activated cell sorter is a flow cytometry instrument designed for sorting and analyzing cells. It utilizes fluorescent labeling and laser excitation to detect and separate individual cells based on their physical and chemical properties.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (2 mentions)
Nextseq, by Illumina (2 mentions)
Kapa library preparation kit, by Roche (2 mentions)
Amaxa kit r, by Lonza (1 mentions)
Cd4 apc clone okt4, by BioLegend (1 mentions)
Anti human cd66b percpcy5.5 clone g10f5, by BioLegend (1 mentions)
Aria 2, by BD (1 mentions)
6 protocols using aria 2 fluorescence activated cell sorter
1
Fluorescent Protein Expression in NK92 Cells
2
Transcriptome Analysis of Lung Cell Types
3
MDSC Isolation and Characterization
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Peripheral blood was collected in sterile heparinized tubes from each patient, and PBMC, obtained by Ficoll–Hypaque gradient centrifugation, were analyzed by flow cytometry. The following anti-human fluorescence conjugated antibodies and their corresponding isotype controls used were as follows: anti-human CD11b-APC-cy7 (clone ICRF44, BD), anti-human HLA-DR-PE (clone G46-6, BD), anti-human CD14-FITC (clone 63D3, Biolegend), and anti-human CD66b-Percpcy5.5 (clone G10F5, Biolegend), FVD (BD), CD3-PE-cy7 (clone HIT3a, Biolegend), CD4-APC (clone OKT4, Biolegend), CD8-PB (clone RPA-T8, Biolegend). We used FVD to discriminate dead cells, and MDSC were classified into two subsets, G-MDSC (CD11b+ HLA-DRlow/− CD66b+) and M-MDSC (CD11b+ HLA-DRlow/CD14+). Data were analyzed with FlowJo 10.0 software package (Treestar Inc., Ashland, OR, USA).
For flow cytometric sorting, an Aria II fluorescence activated cell sorter (BD, Mountain View, CA, USA) was used. The strategy for MDSC sorting was CD11b+ HLA-DRlow/− cells from live PBMC. Depletion of MDSC was performed by harvesting the remaining PBMC after MDSC sorting.
For flow cytometric sorting, an Aria II fluorescence activated cell sorter (BD, Mountain View, CA, USA) was used. The strategy for MDSC sorting was CD11b+ HLA-DRlow/− cells from live PBMC. Depletion of MDSC was performed by harvesting the remaining PBMC after MDSC sorting.
4
Purification and RNA-seq of Lung Cell Types
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Lungs were collected, digested, processed into single cell suspensions, and stained with fluorochrome conjugated antibodies as described in a protocol by Nakano et al. 2018.45 (link) Immune cells (CD45+), epithelial cells (CD45-EpCAM+CD31-), and endothelial cells (CD45-EpCAM-CD31+) were purified using the Aria II fluorescence activated cell sorter (BD Bioscience) and lysed in 350μL of Buffer RLT with β-ME (Qiagen). RNA was extracted using the RNeasy Mini kit (Qiagen) with on-column DNase digestion. RNAseq libraries were prepared using 6ng of RNA and the Kapa library preparation kit (Roche) and sequenced at 2x75bp on NextSeq (Illumina). Sequencing data was aligned to Mus Musculus Reference Genome (version GRCm38).
For analysis, gene-level counts were normalized using the DESeq2 package,46 (link) filtered to remove those with a median expression less than 32, and converted to log2. For use with Reactome pathways, mouse genes were converted to human homologs in the human reference genome (version GRCh38.p13), with expression for human genes with multiple mouse homologs computed via mean expression. Enrichment analysis was performed via the fgsea package, using genes ranked by Adar1ΔCd11c vs control FC in each cell type. All code was written in R (v4.1.2).
For analysis, gene-level counts were normalized using the DESeq2 package,46 (link) filtered to remove those with a median expression less than 32, and converted to log2. For use with Reactome pathways, mouse genes were converted to human homologs in the human reference genome (version GRCh38.p13), with expression for human genes with multiple mouse homologs computed via mean expression. Enrichment analysis was performed via the fgsea package, using genes ranked by Adar1ΔCd11c vs control FC in each cell type. All code was written in R (v4.1.2).
5
Apoptosis Induction in A549 Cells
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Induction of apoptosis in Α-MANGOSTIN -treated A549 cells was monitored by AnnexinV/PI double staining, using BD Fluorescence Activated Cell Sorter (ARIA II). Cultured A549 cells (1 × 104 cells/ml) were treated with varying concentrations of α-mangostin (0–10 μM) for 24 h. Induction of apoptosis was determined by flow cytometry using Annexin V-FITC apoptosis kit (Cayman Chemicals) following the protocol supplied by the manufacturers.
6
Apoptosis, ROS, and Mitochondrial Dynamics
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For detection of apoptosis, treated cells (2.5×106) werewashed with 1X PBS, stained doubly with FITC conjugated AnnexinV antibody and PI (Propidium Iodide) according to manufacturer’s instructions(Cayman Chemicals) and then subjected to flow-cytometric analysis [BD Fluorescence Activated Cell Sorter (ARIA II), San Jose, CA] using FACS Diva software.
IntracellularROS generation was measured by changes in fluorescence intensity of H2DCFDA (excitation480nm, emission 530 nm) by flow-cytometry using cells treated with H2DCFDA (10 μM) in dark for 30 min at 37°C.
The changes inMitochondrial Membrane Potential (MMP) of treated cells with respect to the controls (1x106)were detected by exposing cells to different concentrations of WA or equal amount of DMSO (vehicle) for 24h, or 10 mM H2O2 for 15 min, followed by incubating themwith TMRE (100 nM) for 30 min at 37°C in dark. After washing with PBS, fluorescence intensities(PE-A, 575 nm) of 10,000 cells wereanalyzed by flow cytometry(BD FACS, ARIA II) using FACS Diva software.
IntracellularROS generation was measured by changes in fluorescence intensity of H2DCFDA (excitation480nm, emission 530 nm) by flow-cytometry using cells treated with H2DCFDA (10 μM) in dark for 30 min at 37°C.
The changes inMitochondrial Membrane Potential (MMP) of treated cells with respect to the controls (1x106)were detected by exposing cells to different concentrations of WA or equal amount of DMSO (vehicle) for 24h, or 10 mM H2O2 for 15 min, followed by incubating themwith TMRE (100 nM) for 30 min at 37°C in dark. After washing with PBS, fluorescence intensities(PE-A, 575 nm) of 10,000 cells wereanalyzed by flow cytometry(BD FACS, ARIA II) using FACS Diva software.
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