Single cell suspensions were incubated with an antibody cocktail containing APC-anti-CD11b, FITC-anti-CD47 and PE-Cy7-anti-Ly6G for 30 min at room temperature. For blockade of Fc receptors on monocyte/macrophages and neutrophils during flow cytometric analysis, the cells were pre-incubated with 0.5 µg purified rat anti-CD16/CD32 for 5 min on ice prior to staining with antibody cocktail. Apoptotic cells were detected by PE-Annexin V apoptosis detection kit (BioLegend; cat. no. 640934), according to the manufacturer's recommendations. All stained cells were analyzed on a BD FACSAria™ III instrument (BD Biosciences) and FlowJo software, v. 8.8.4 (FlowJo LLC).
Facsaria 3 instrument
Manufactured by BD
Sourced in United States
The FACSAria III is a flow cytometry instrument designed for cell sorting and analysis. It is capable of detecting and sorting multiple fluorescent signals simultaneously. The instrument is equipped with multiple lasers and detectors to enable high-performance cell sorting and data acquisition.
Automatically generated - may contain errors
Lab products found in correlation
Facsdiva software, by BD (4 mentions)
Cli 095, by InvivoGen (2 mentions)
Anti hamster igg, by Thermo Fisher Scientific (2 mentions)
Paraformaldehyde (pfa), by Merck Group (2 mentions)
Brefeldin a, by Thermo Fisher Scientific (2 mentions)
Anti cd3 ucht1, by BioLegend (2 mentions)
Saponin, by Carl Roth (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Lipopolysaccharides (lps), by Merck Group (2 mentions)
Staining buffer, by BD (2 mentions)
Anti cd86 af700 clone gl1, by BD (2 mentions)
F4 80 bm8, by BioLegend (2 mentions)
Pe annexin 5 apoptosis detection kit, by BioLegend (1 mentions)
Ls003126, by Worthington (1 mentions)
D5025, by Merck Group (1 mentions)
C7352, by Merck Group (1 mentions)
4d nucleofector x unit, by Lonza (1 mentions)
Fta sample collection kit, by American Type Culture Collection (1 mentions)
Perc cy5 anti f4 80, by BD (1 mentions)
Anti cd45 pe, by BD (1 mentions)
52 protocols using facsaria 3 instrument
1
Multicolor Flow Cytometry of Myeloid Cells
2
Cell Cycle Analysis by PI Staining
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell cycle distribution was determined by DNA staining with PI (#550825, BD Pharmingen, San Diego, CA, USA). A total of 1 × 106 cells was collected and fixed in 70% ethanol. Cell pellets were suspended in PI/RNase staining buffer and incubate for 15 min at room temperature before analysis. The percentages of cells in different phases of the cell cycle were measured using BD FACSAria™ III instrument.
3
Astrocyte Purification from Optic Nerve Head
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Astrocytes were purified from the optic nerve head of Gfap-tdTomato and Spp1GFPfl/flGfapCre mice. Briefly, the optic nerve heads were treated with papain (0.6 mg/ml; LS003126; Worthington) and L-cysteine (0.012 mg/ml; C7352; Sigma-Aldrich) for 15 min at 37°C in Ca2+- and Ma2+-free HBSS solution (14185-052; Gibco). After the incubation, tissues were centrifuged at 953g for 5 min and the papain/HBSS solution was removed. Tissues were resuspended in horse serum (10%, 26050-088; Gibco) and DNase I (60 U/ml; D-5025; Sigma-Aldrich) in HBSS and were triturated with a heat-polished Pasteur pipet (TW150-4; World Precision Instruments), and the tissue was completely dissociated. Dissociated cells were centrifuged, resuspended in HBSS, and passed through a 35-μm cell strainer. Astrocytes were identified by tdTomato fluorescence and sorted directly into a collection medium using a BD FACSAria III instrument (BD Biosciences). Astrocytes sorted by FACS were centrifuged at 1,000g for 10 min and were used for RNA extraction.
4
CRISPR-Cas9 Knockout of B7-H3 in LM7 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
B7-H3−/− LM7 cells (LM7KO) were generated using CRISPR-Cas9 technology. Briefly, 400,000 LM7 cells were transiently transfected with precomplexed ribonucleoproteins (RNPs) consisting of 150 pmol of chemically modified single guide RNA (sgRNA) (5′-GAUCAAACAGAGCUGUGAGG-3′, Synthego) and 35pmol of Cas9 protein (St. Jude Protein Production Core) via nucleofection (4D-Nucleofector X unit; Lonza, Switzerland) using solution P3 and program DS-150 in a small (20-μL) cuvette according to the manufacturer’s recommended protocol. A portion of the pool of cells was harvested 3 days after nucleofection and verified to contain the desired modification via targeted deep sequencing and analysis with CRIS.py.45 (link) Post-nucleofection LM7KO cells were stained with B7-H3 antibody (clone 7-517; Becton Dickinson, Franklin Lakes, NJ, USA) and sorted on the B7-H3-negative population using a BD FACSAria III instrument. This sorting step was repeated one additional time to produce a final LM7KO product. After KO and flow sorting, LM7KO cells were authenticated by short tandem repeat (STR) profiling using the service of the American Type Culture Collection (FTA sample collection kit; ATCC, Manassas, VA, USA).
5
PRRSV Antigen Presentation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Sorted cDCs, CD14+ cells and PBMCs were stimulated with PRRSV at an moi of 0.1 in 96-well plates seeded with 1 × 104 cells per well. They were then incubated for 24 h at 37 °C and washed with RPMI-1640 medium to eliminate the remaining virus. Sorted T cells were labeled with carboxyfluorescein succinimidyl ester (CFSE). Briefly, the cells were stained with CFSE for 15 min (mixing every 5 min), and 1 mL of FBS was then added to eliminate the CFSE excesses. The T cells were centrifuged at 1600 rpm for 10 min at 25 °C, cocultured with the cDCs or monocytes at a ratio of 1:10 for 96 h and analyzed with the BD FACS ARIA III instrument.
6
Multicolor Flow Cytometry Profiling of Lung Immune Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
0.3 × 106 cell suspension from lung digests or BAL were incubated with antibody cocktail containing FITC-anti-CD206, PE-anti-CD45, PerC-Cy5-anti-F4/80, PE-Cy7-anti-Ly6G, APC-Cy7-anti-CD11b, BV421-anti-Siglec F (also known as CD170). All antibodies were purchased from BD Biosciences (Franklin Lakes, NJ), BioLegend and eBiosciences (San Diego, CA). The cells were stained with fluorescence-minus-one (FMO) antibody cocktail for negative staining control. After incubation with antibodies in PBS supplied with 3% FBS for 40 min at room temperature, the cells were then washed with PBS for 2 times and analyzed on BD FACSAria™ III instrument and BD FACSDiva™ software (BD Biosciences, San Jose, CA) within 48 h. All data was analyzed using FlowJo software, version 8.8.4 (Tree Star Inc.).
7
Immune Cell Profiling by Flow Cytometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The 0.3 × 106 cell suspensions of lung digests or BAL were incubated with an antibody cocktail containing PE-anti-CD45, PerC-Cy5-anti-F4/80, PE-Cy7-anti-Ly6G, APC-Cy7-anti-CD11b, and BV421-anti-Siglec-F (BD Biosciences, Franklin Lakes, NJ, USA; eBiosciences, San Diego, CA, USA). After incubation with the antibody cocktail for 40 min at room temperature, the cells were washed twice with PBS. The stained cells were analyzed by a BD FACSAria™ III instrument and BD FACSDiva™ software (BD Biosciences, San Jose, CA, USA). All data were analyzed using FlowJo software, version 8.8.4 (Tree Star Inc., Ashland, OR, USA).
8
Lung Adenocarcinoma Single-Cell Isolation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All the included patients were pathologically diagnosed with lung adenocarcinoma. About 1cm3 of tumor tissue and tumor adjacent tissue during curative surgery were collected and dissociated into single-cell suspensions by Tumor Dissociation Kit (130-096-730, Miltenyi). Cell suspensions were added with ACK lysis buffer (A10492-01, Gibco) to lyse the remnant RBC, and treated with Dead Cell Removal Kit (130-090-101, Miltenyi), then filtered through the 40-μm plastic mesh (Falcon). Cell suspensions were stained with DAPI for viability, and living cells were sorted with a BD FACSAria III instrument.
9
Cell Surface Staining and Flow Cytometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After surface staining, cells were resuspended in BD Cytofix/Cytoperm (BD Bioscience) and incubated for 20 min at 4 °C. Cells were washed in Perm/Wash Buffer (BD Bioscience), incubated with the relevant mAb for 20 min at 4 °C, and washed again twice. Data were acquired with a BD FACSAria™ III instrument.
10
Multiparametric Flow Cytometry Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PBMCs stimulated overnight from day 10 were harvested and washed for staining assays. First, cells were incubated with reagents from a live/dead fixable viability kit (catalog number 423102, BioLegend) at room temperature in the dark for 15 min. The cells were then washed again, and surface staining was performed with the following antibodies: APC/Cy7 anti-human CD3, FITC anti-human CD4, and Pacific blue anti-human CD8. Cells were subsequently fixed and permeabilized using a Cytofix and Perm kit (BD Bioscience) and stained with the following intracellular cytokine antibodies: phycoerythrin (PE) anti-human TNF-α and APC anti-human IFN-γ. Finally, the cells were resuspended in 200 μL fluorescence-activated cell soring buffer and analyzed on a BD FACSAria III instrument. Data were evaluated using FlowJo software.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!