Analyses were performed on a BD FACSAria III SORP (Special Order Research Product) or LSR Fortessa (both BD Biosciences, San Diego, CA) and the data analyzed with FlowJo version 9.4.11 (Tree Star, Ashland, OR).
Facsaria 3 sorp
Manufactured by BD
Sourced in United States
The FACSAria III SORP is a high-performance cell sorter designed for advanced flow cytometry applications. It offers robust and reliable performance for cell sorting, cell analysis, and sample preparation.
Automatically generated - may contain errors
Lab products found in correlation
Lsrfortessa, by BD (2 mentions)
Robosep, by STEMCELL (2 mentions)
Allophycocyanin tcrβ, by Thermo Fisher Scientific (2 mentions)
Fitc conjugated mouse anti cd150a pdgfra antibody, by Thermo Fisher Scientific (2 mentions)
Collagenase type 2, by Merck Group (2 mentions)
Lsrfortessa sorp, by BD (1 mentions)
Red blood cell lysis buffer, by Merck Group (1 mentions)
Fitc cd45.1 a20, by BD (1 mentions)
Heparinized capillary tube, by Thermo Fisher Scientific (1 mentions)
Cd45.1 apc a20, by BioLegend (1 mentions)
Cd8 pecy7 53 6.7, by Cytek Biosciences (1 mentions)
B220 pe cy7, by Cytek Biosciences (1 mentions)
Cd4 pecy7 rm4 5, by BD (1 mentions)
Pe anti mouse cd8a, by BD (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
Anti cd16 cd32, by BD (1 mentions)
Fitc anti mouse cd4, by BD (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Collagenase 2, by Worthington (1 mentions)
Doxycycline, by Merck Group (1 mentions)
20 protocols using facsaria 3 sorp
1
Flow Cytometric Analysis with BD Instruments
2
Flow Cytometry Isolation of ESCs
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Cells were detached using 0.25% trypsin and resuspended in PBS containing 2.5% ESC-grade and 5 mM EDTA (pH 8). Suspended cells were then analyzed by the BD LSRFortessaSORP flow cytometry system using DiVa software. For downstream analysis on sorted ESCs, cells were sorted according to the fluorescence intensity of eGFP into PBS using BD FACSAria III SORP with a 85-µm nozzle. Data analysis was performed with FlowJo software (version 10.5.3).
3
Multicolor Flow Cytometry Protocol for Murine Immune Cell Analysis
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PB was collected from the retro-orbital plexus in heparinized capillary tubes (Fisherbrand, Pittsburgh, PA) and lysed in red blood cell lysis buffer (Sigma-Aldrich, St. Louis, MO). Cells were stained with the following antibodies: CD45.1-APC (A20) (Biolegend, San Diego, CA) or CD45.1-FITC (A20) (BD Biosciences, San Diego, CA), B220-PECy7 and CD8-PECy7 (53–6.7) (Tonbo Biosciences, San Diego, CA), CD45.2-V500 (104), B220- PerCPCy5.5 (RA3-6B2), Gr1-PerCPCy5.5 (RB6-8C5), Cd11b-PerCPCy5.5 (M1/70) and CD4-PECy7 (RM4-5) (BD Biosciences, San Diego, CA). All antibodies were used at 1:200 dilution. 4’,6-diamidino-2-phenylindole (DAPI) staining was used to gate live events. Analysis was performed on a LSR Fortessa and a BD FACSAria III SORP (Special Order Research Product, which contains the following LASERs: 405 nm, 445 nm, 488 nm, 562 nm, and 640 nm) (both BD Biosciences, San Diego, CA) and the data analyzed with FlowJo version 9.4.11 (Tree Star, Ashland, OR). To discern the four Confetti colors: the filter arrangements for each LASER were: CFP, 445 nm LASER—470/24 band-pass filter (BP); GFP, 488 nm LASER—515/20 BP and 505 long-pass filter (LP); YFP, 488 nm LASER—545/10 BP and 525 LP; RFP, 562 nm LASER—610/20 BP and 600 LP48 (link).
4
Isolation and Analysis of T Cell Subsets
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Single-cell lymphocyte solutions were prepared from organs as indicated and resuspended in staining buffer containing 5% FBS in HBBS. Pre-enrichment of splenic lymphocytes was performed using PE-labeled CD8+ T cell magnetic bead positive selection according to the manufacturer instructions (RoboSep, STEMCELL Technologies). Splenic and brain-resident lymphocytes were identified using mAbs against the following Ags, all from BioLegend unless otherwise noted: PE-CD8α (MAR1), FITC-CD4 (RM4–5), BV711-CD11b (M1/70), BV421-CD45.2 (1D4), allophycocyanin/Cy7-CD45.2 (1D4), and allophycocyanin-TCRβ, (H57–597; eBio science). Samples were sorted on an FACSAria III SORP (BD Biosciences) equipped with violet (403 nm, 100 mW), blue (488 nm, 100 mW), yellow-green (561 nm, 50 mW), and red (638 nm, 150 mW) lasers, then analyzed with FlowJo (Tree Star). Pre- and postsort purity of CD8+ T cells are shown in Fig. 1A .
5
Isolation and Analysis of T Cell Subsets
6
Isolation and Treatment of Murine Thymic DP Cells
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Three-week-old C57BL/6 mice were used for isolation of thymus. Thymi were used for the preparation of single cell suspension and subjected to Fc receptor blocking using the purified anti-CD16/CD32 (Clone 2.4G2, BD Biosciences). Thymocytes were then surface stained using the following fluorochrome tagged antibodies: FITC anti-mouse CD4 (Clone GK1.5, BD Biosciences); PE anti-mouse CD8a (Clone 53–6.7, BD Biosciences). CD4+CD8+ DP thymocytes were FACS sorted using FACS Aria III SORP (BD biosciences). Sorted DP thymocytes were cultured in the presence or absence of different concentrations of MG-132 (Calbiochem), and Cycloheximide (Sigma-Aldrich) in RPMI-1640 media supplemented with 10% FBS and penicillin-streptomycin for 4 h. After incubation, cells were harvested and used for western blotting.
7
Fluorescent Activated Nuclear Sorting
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Flow cytometry analysis and FANS were performed using a BD FACSAria III SORP equipped with four lasers (405, 488, 561, and 640 nm) and a 70 µm nozzle. GFP expression was detected using the blue laser and a 530/30 BPfilter, whereas DAPI was detected using the violet laser and a 450/50 BP filter. Prior to sorting, 10,000 total events were recorded, and a gating strategy was applied: first, nuclei were gated according to their forwardand side-scatter properties (FSC-A/SSC-A), followed by doublet exclusion using SSC-A and SSC-W. Nuclei were then gated according to their DAPI expression. GFP expression was used as a sorting gate. Sorted nuclei were snap-frozen in a mixture of dry ice and 100% ethanol and stored at -80 °C before their respective RNA isolation extractions.
8
Isolation and Sorting of Skin Mesenchymal Progenitor Cells
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Mouse dorsal skin was minced and digested with 0.2% collagenase II (CLSII Worthington Biochemicals) in PBS supplemented with 20% FBS for 60 min at 37°C. After washing with PBS/20% FBS followed by PBS, the digested skin tissue was treated with 0.05% trypsin-EDTA (GIBCO) for 10–15 min at 37°C. The cell suspension was spun down at 300 g for 10 min. Mononuclear cells were then incubated with FcR (CD16/32) antibody and stained with CD45, TER119, CD31, CD44, CD140a, and SCA1 for 15 min at 2–8°C. Dead cells were excluded by propidium iodide (PI) staining. For sorting native skin MPCs, hematopoietic and endothelial populations were firstly excluded (CD45−TER119−CD31−) and Ebf2 gate was then defined based on fluorescence minus one (FMO) using skin mononuclear cells from a non-transgenic mouse. Skin Ebf2+ and Ebf2− cells were sorted on a FACS Aria III Sorp (BD Biosciences). See Table S1 for detailed antibody information.
9
FACS Sorting of Zebrafish PGCs
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The vasa:eGFP line (Krøvel and Olsen, 2002 (link)) was used for FACS of PGCs at different time points. Embryos were collected and incubated with TrypLE Express for 40 to 70 min, killed on ice and gently pipetted up and down with a glass pipet and/or a 200 µl low-retention pipet tip. After visual inspection, cell suspension was separated from trunks using a 100 µm sieve. Following another 5-15 min of digestion, FCS was added to 10% of total volume. Cells were spun down at 500 g for 5 min at room temperature, washed with PBS, resuspended in PBS with 2% FCS, put on ice and immediately subjected to FACS using a 85 µm nozzle on a BD FACSAria III SORP (Becton Dickinson). Between 1000 and 2500 cells were sorted directly into Trizol. RNA from sorted PGCs and whole embryos was extracted with Trizol and stored in MQ at −80°C until library preparation was carried out.
10
Inducible Expression of DDX41 Variants in AML Cells
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Expression of N-terminally GFP-tagged DDX41 WT, L237F + P238T or R525H in OCI-AML3 cells was induced with 3 µg/ml doxycycline (Sigma-Aldrich). 72 h after induction, cells were spun down and washed twice with PBS. After re-suspending cells in PBS, they were sorted by FACS using a 100 µM nozzle on a BD FACSAria III SORP (Becton Dickinson) in purity precision mode with FACSDiva software version 8.0.2. Cells of interest were identified via FSC-A/SSC-A. Subsequently, doublets were excluded via FSC-A/FSC-H and dead cells were excluded by DAPI staining (0.5 µg/ml final concentration) using a 405 nm laser and 450/50 BP. GFP cutoff was set according to non-expressing cells. Sorting based on GFP was achieved using the 488 nm laser and 530/30 band pass filter. Roughly 500,000 cells were sorted directly into fresh a-MEM containing 20% FBS and further cultured until subsequent experiments.
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