Draining lymph node cells were collected for further analysis at the end of CIA as described above. For regulatory T cell analysis, the cells were surface-stained with antibodies against CD4 and CD25, followed by intracellular staining with anti-Foxp3 antibody (eBioscience; San Diego, CA). For the Th1 and 17 cells analysis, the lymph node cells were stimulated with PMA (50 ng/ml; Sigma-Aldrich, St. Louis, MO, USA) plus ionomycin (1 μg/ml; Sigma-Aldrich, St. Louis, MO, USA) for 5 h in culture medium in the presence of GolgiStop (BD Biosciences, San Jose, CA, USA), then surface-stained with anti-CD4 antibody followed by intracellular staining with anti-IL-17 and anti-IFN-γ antibodies (eBioscience; San Diego, CA). The cells were analyzed on an LSRII flow analyzer (BD Biosciences, San Jose, CA, USA) using FlowJo (Tree Star, Ashland, OR) (Qiao et al. 2012 (link); Ying et al. 2010 (link);).
Lsrii flow analyzer
Manufactured by BD
Sourced in United States
The LSRII flow analyzer is a laboratory instrument designed for the analysis and enumeration of cells, particles, and other biological samples. It utilizes laser-based technology to detect and measure various physical and fluorescent characteristics of individual cells or particles as they pass through a flow cell. The LSRII provides high-speed, multiparameter analysis capabilities for a wide range of applications in fields such as immunology, stem cell research, and cell biology.
Automatically generated - may contain errors
Lab products found in correlation
Anti foxp3 antibody, by Thermo Fisher Scientific (1 mentions)
Anti il 17, by Thermo Fisher Scientific (1 mentions)
Anti ifn γ antibody, by Thermo Fisher Scientific (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Golgistop, by BD (1 mentions)
Counting beads, by Thermo Fisher Scientific (1 mentions)
Live dead violet, by Thermo Fisher Scientific (1 mentions)
Pd l1, by BioLegend (1 mentions)
Hla dr, by BioLegend (1 mentions)
Cd66b, by BioLegend (1 mentions)
Cd9 1, by Thermo Fisher Scientific (1 mentions)
Lyse fix buffer, by BD (1 mentions)
Rat serum, by Jackson ImmunoResearch (1 mentions)
Mouse serum, by Jackson ImmunoResearch (1 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions)
3 protocols using lsrii flow analyzer
1
Regulatory T Cell and Th17 Analysis
2
Monocyte Phenotyping and Intracellular NO Analysis
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Monocytes from blood (pre-transmigration and post-transmigration) were stained as described before [8 (link)], with the following modifications. Antibodies were directed against CD33 (Biolegend, San Diego, CA, USA, cat# 366614), CD36 (Biolegend, cat# 336204), CD47 (Biolegend, cat# 323114), CD63 (Biolegend, cat# 353026), CD66b (Biolegend, cat# 392904), CD91 (Invitrogen, cat# 46091942), CD172a (Biolegend, cat# 372106), HLA-DR (Biolegend, cat# 307626), PD-1 (Biolegend, cat# 329952), and PD-L1 (Biolegend, cat# 329738). Viability was determined based on staining with Live/Dead Violet (ThermoFisher Scientific, Wlatham, MA, USA, cat# L34958). Intracellular NO was measured with the cell-permeable probe diaminofluorescein-2-diacetate, as detailed before [5 (link)]. Samples were incubated with antibodies and viability or NO probe in the dark, at 4 °C for 30 min, washed with PBS-EDTA, and centrifuged at 400× g at 4 °C for 10 min, after which the supernatant was removed and samples were fixed overnight at 4 °C in Lyse/Fix buffer (BD Biosciences, San Jose, CA, USA, cat# 558049). Samples were resuspended in 300 µL PBS plus 10 µL counting beads (ThermoFisher Scientific, cat# C36950) to enable absolute cell counting during acquisition on a LSRII flow analyzer (BD Biosciences).
3
Multicolor flow cytometry for immune cell profiling
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Freshly prepared cell suspensions were blocked with 1% mouse serum (Jackson ImmunoResearch), 1% rat serum (Jackson ImmunoResearch), and 2% mouse FcR Blocking Reagent (Miltenyl) for 15 mins at 4 o C. Fluorophore-conjugated primary antibodies against cell surface antigens were added to the cell suspensions and incubated for 30 mins at 4 o C. Cells were then washed with cold PBS and stained with LIVE/DEAD UV (Invitrogen) according to manufacturer's instructions. Cells were then washed with cold FACS buffer and fixed with Foxp3/Transcription Factor Staining Buffer Set (eBioscience) according to manufacturer's protocol. Fixed cells were again blocked with 1% mouse serum, 1% rat serum, and 2% mouse FcR Blocking Reagent for 15 mins at 4 o C and then stained with fluorophore-conjugated primary antibodies against intracellular antigens. Cells were then washed, and data were acquired on a LSR II Flow Analyzer (BD). Details on the antibodies used are provided in Supplementary Table S4. Data acquired were analyzed by using FlowJo software (BD).
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