Fentanyl-reactive B cells were analyzed on an LSR II instrument and isolated using an ARIA II cell sorter (BD Bioscience). Cells were stained using fentanyl-PE (1:1000; 1 μM stock), a decoy-PE-AF647 (1:50; 1 mM stock)49 (link),50 (link),95 (link) and the following antibodies: rat anti-mouse CD19-BV412 (1:100, Biolegend), rat anti-mouse IgG1-BV650 (1:100, Biolegend), rat anti-mouse CD138-BV510 (1:300, Bio-legend), rat anti-mouse GL-7-FITC (1:1000, BD Pharmingen), rat anti-mouse CD38-PE-Cy7 (1:400, Biolegend), goat anti-mouse IgM-biotin (1:400, Jackson), rat anti-mouse IgD-APC-Cy7 (1:1000, Biolegend), streptavidin-BV785 (1:400, Biolegend). The LIVE/DEAD™ Fixable Blue Dead Cell Stain Kit (1:1000, Invitrogen) was included in all stainings to exclude dead cells. The data were analyzed using FlowJo v10 software. For single-cell sorting, fentanyl-reactive B cells were defined as live fentanyl + decoy-CD19+ and sorted into 384-well plates containing lysis buffer using an ARIA II cell sorter (BD Bioscience and using the index-sort function).
Aria 2 cell sorter
Manufactured by BD
Sourced in United States, Germany
The Aria II cell sorter is a laboratory instrument designed for the separation and isolation of individual cells from a heterogeneous cell population. It utilizes flow cytometry technology to precisely sort and collect specific cell types based on their physical and fluorescent characteristics.
Automatically generated - may contain errors
Lab products found in correlation
Lsrii flow cytometer, by BD (7 mentions)
Facsdiva software, by BD (6 mentions)
Microamp optical 96 well reaction plate, by Thermo Fisher Scientific (3 mentions)
Fortessa, by BD (3 mentions)
Live dead fixable blue dead cell stain kit, by Thermo Fisher Scientific (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Nucleospin rna xs kit, by Macherey-Nagel (2 mentions)
One step rt pcr reaction mix, by Thermo Fisher Scientific (2 mentions)
Biotin conjugated rat antibodies specific for mouse cd45, by Thermo Fisher Scientific (2 mentions)
Anti rat igg dynabeads, by Thermo Fisher Scientific (2 mentions)
Brdu flow kit, by BD (2 mentions)
Live dead fixable yellow dead cell stain, by Thermo Fisher Scientific (2 mentions)
Magnetic dynabeads, by Thermo Fisher Scientific (2 mentions)
Fixation permeabilization buffer, by Thermo Fisher Scientific (2 mentions)
Accutase, by Thermo Fisher Scientific (2 mentions)
Cell strainer, by BD (2 mentions)
Apc conjugated anti mouse epcam, by Thermo Fisher Scientific (2 mentions)
Cytofix cytoperm kit, by BD (2 mentions)
Collagenase a, by Roche (2 mentions)
Quick gdna miniprep kit, by Zymo Research (2 mentions)
123 protocols using aria 2 cell sorter
1
Fentanyl-reactive B Cell Isolation
2
Single-cell RNA-seq analysis of homeostasis regulators
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For homeostasis experiments, K5VP16;Pparg;mTmG and K5;mTmG single-cell suspensions were filtered through a 70 μm filter (Fisherbrand cat#22363548) and then sorted on a BD Aria II Cell Sorter using a 130 μm nozzle aperture and 13 psi pressure to collect GFP-positive cells. Gating strategy was performed on BD FACSDiva Software v 8.0. Cells were then centrifuged at 500 × g for 30 min at 4°C. The supernatant was discarded, and the pellet was processed for total RNA extraction. Samples with a RIN (regulation identification number) >8 were used for RNA-seq. The libraries were prepared using the SMART-Seq® v4 Ultra® Low Input RNA Kit for Sequencing (TaKaRa) followed by Nextera XT (Illumina), both according to manufacturer’s instructions. They were sequenced to a targeted depth of 40 M 2 × 100 bp reads on a NovaSeq 6000 (Illumina). Differential expression analysis was performed by reading kallisto counts files into R using the R packages tximport (v.1.10.1) and biomaRt (v.2.34.2), and running DESeq2 (v.1.18) to generate log fold change values and p values between the two experimental groups. The heatmap and PCA plots were visualized after transforming the counts using VST (variance stabilizing transformation). Gene set analysis by ConsensusPathDB (Kamburov, A. et al. 2013) was used to identify significantly changed pathways.
3
Murine Hematopoietic Stem Cell Isolation
4
Multiparametric Flow Cytometry Analysis
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Cells were labelled with flow antibodies for 15 minutes in the dark in PBS containing 2% BSA. Labelled cells were washed twice and resuspended in PBS containing 2% BSA. The prepared samples were analyzed using an LSR-II flow cytometer (BD Bioscience) or sorted using an Aria II cell sorter (BD Bioscience). Anti-CD19-PeCy7 (561739), anti-CD3-PeCy7 (552774), anti-NK1.1-BV421 (562921), anti-NKp46-FITC (560756), anti-NKp46-AF647 (560755), anti-CD49b-PE (553858), anti-CD49a-PerCP-Cy5.5 (564862), anti-CD62L-APC (553152), anti-T-bet-APC (561264), anti-CD45.2-AF700 (560693), anti-CD45.2-FITC (553772), anti-Gata3-AF647 (560068), anti-RORγt-PE (562607), anti-CD127-V450 (561205), anti-CD117-PE (553869), anti-LPAM-1-APC (562376), anti-Flt3-BV421 (566292), anti-Ly-6A/E-APC (565355), and anti-CD122-PE (553362) were purchased from BD Bioscience. Anti-IFN-γ-AF-700 (505823) and anti-CD25-Pacific Blue (102022) were purchased from Biolegend. Anti-CD253-APC (17-5951-82), anti-Eomes-PE (12-4875-82), and anti-CD127-PerCP-Cy5.5 (45-1271-80) were purchased from eBioscience.
5
Isolation and Treatment of Cancer-Associated Fibroblasts
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Transplanted tumors were harvested at the indicated time points to prepare single-cell suspensions as previously described. After red blood cell lysis and blocking of Fc receptors, cells were stained with anti-PDGFRα and anti-F4/80 antibodies (Biolegend) at a 1:100 dilution for 30 min on ice. CAFs (PDGFRα+F4/80-) were sorted by Fluorescence Activated Cell Sorting (FACS) on an Aria II cell sorter (BD Biosciences) 15 . Sorted CAFs were suspended in RNA lysis buffer (Invitrogen) or seeded in 24-well plates in DMEM containing 10% FBS and 100 units/ml penicillin/streptomycin. Subsequently, non-adherent cells were removed by extensive washing with PBS and adherent cells were treated with recombinant mouse Fgl2 (1 µg/ml or 5 µg/ml, R&D Systems) for further analysis.
6
Isolation and FACS of Retinal Cells
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We used Rhoi75Cre and HRGPCre lines in which the Z/EG transgene was introduced. These animals express the CRE reporter enhanced green fluorescent protein (eGFP) in rods and cones, respectively. Retinas were isolated and enzymatically digested with trypsin (0.25%) for up to 15 min at 37°C. Subsequently, they were triturated with a plastic pipette tip in a deoxyribonuclease I solution (400 U/ml) to obtain single-cell suspension. During the whole procedure, the retinas were incubated in Hepes-buffered Ames’ medium under room lights. Cell sorting was performed with an Aria II cell sorter (BD Biosciences). Selection was based on fluorescence and forward scatter. Collected cells were lysed and stored in the lysis buffer of a NucleoSpin RNA XS kit (Macherey-Nagel) until further treatment.
7
Single-cell MAIT transcriptomic profiling
8
Isolation of Osx1-Expressing Bone Cells
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The tibiae and femurs were dissected from mice immediately after death. Total bone marrow cells were flushed from the bones, using a 23‐gauge needle and syringe, into ice‐cold FACS buffer containing CaCl2‐ and MgCl2‐free 1X PBS (Thermo Fisher Scientific, Carlsbad, CA, USA) and 2% FBS. Cells from individual mice in each group were centrifuged at 450 g for 6 min at 4 °C. After the red blood cells were removed with RBC lysis buffer (0.9% NH4Cl with 20 mm Tris base, pH 7.4), bone marrow cells were suspended in ice‐cold FACS buffer. Cells were then incubated with biotin‐conjugated rat antibodies specific for mouse CD45 (eBioscience, San Diego, CA, USA; 14‐0451, 1:100). The labeled hematopoietic cells were depleted 3 times by incubation with anti‐rat IgG Dynabeads (Invitrogen, Grand Island, NY, USA) at a bead:cell ratio of approximately 4:1. Cells binding the Dynabeads were removed with a magnetic field. The negatively isolated CD45− cells were washed twice and suspended with ice‐cold FACS buffer at 1–2 × 106 cells mL−1. Osx1‐TdRFP+ cells were sorted in an Aria II cell sorter (BD Bioscience, San Jose, CA, USA) using the PE‐A fluorochrome gate.
9
Isolation and Purification of Hematopoietic Stem Cells
10
Isolating Ma-Mel-36 Subpopulations by Flow Cytometry
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The following directly labelled antibodies were used for staining of cellular surface markers: anti-HLA-ABC-APC (eBiosciences, clone W6/32; 1 μl), anti-CD54-PE (Beckmann Coulter, clone 84H10; 2.5 μl), anti-PD-L1-PE (Biolegend, clone 29E2A3; 5 μl) and anti-HLA-DR-PC7 (Beckmann Coulter, clone Immun-357; 2.5 μl). After fixation, stained cells were analysed by flow cytometry on a Gallios flow cytometer (Beckmann Coulter) and Kaluza (Beckman Coulter) software, respectively, for data analysis. In order to isolate specific Ma-Mel-36 subpopulations, cells were stained with anti-HLA-DR-PC7 and sorted based on the specific expression of the surface markers by flow cytometry on an Aria II cell sorter and the FACS Diva software (BD Biosciences).
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