ILC2s were cultured in the presence of rm-IL-2 (10 ng/mL) and rm-IL-7 (10 ng/mL). When indicated, ILC2s were incubated with purified mouse C1q (10 μg/mL, Complement Technology) or with 15 μM of the SHP-1 inhibitor, known as Tyrosine Phosphatase Inhibitor 1 (TPI-1, MedChemExpress)39 (link),43 . The BD Biosciences Cytofix/Cytoperm kit was used and followed by intracellular staining with PE anti-p65 (clone 532301, R&D Systems), BV421 anti-AKT (pS473) (clone M89-61, BD Biosciences), PE-Cy7 anti-BCL-2 (clone 10C4, eBioscience) and unconjugated anti-SHP-1 (clone SR41-02, ThermoFisher) followed by Alexa Fluor 647 goat anti-rabbit IgG secondary antibody (Jackson ImmunoResearh). Intranuclear staining was performed using the Foxp3 Transcription Factor Staining Kit (ThermoFisher) according to the manufacturer’s instructions and APC anti-mouse Ki67 (SolA15, ThermoFisher) was used to assess proliferation. The CellTrace Violet Cell Proliferation Kit (ThermoFisher) was also used in some experiments, according to manufacturer’s instructions. In parallel, the levels of IL-5, IL-6, and IL-13 were measured in supernatants using Legendplex multiplex kits (BioLegend) and data were analyzed via the LEGENDplex data analysis software v8.0. GM-CSF was quantified using ELISA kit from BioLegend.
Cytofix cytoperm kit
Manufactured by R&D Systems
Sourced in United States
The Cytofix/Cytoperm kit is a laboratory product designed to enable the fixation and permeabilization of cells for subsequent intracellular staining and flow cytometry analysis. The kit provides the necessary reagents to prepare cells for the detection of intracellular proteins, cytokines, and other cellular components.
Automatically generated - may contain errors
Lab products found in correlation
Cytofix cytoperm kit, by BD (2 mentions)
Tpi 1, by MedChemExpress (1 mentions)
Pe cy7 anti bcl 2 clone 10c4, by Thermo Fisher Scientific (1 mentions)
Foxp3 transcription factor staining kit, by Thermo Fisher Scientific (1 mentions)
Legendplex multiplex kits, by BioLegend (1 mentions)
Celltrace violet cell proliferation kit, by Thermo Fisher Scientific (1 mentions)
Anti cd8 antibody, by R&D Systems (1 mentions)
Anti goat igg pe cy7, by Santa Cruz Biotechnology (1 mentions)
Brefeldin a, by Merck Group (1 mentions)
Cytomics fc500, by Beckman Coulter (1 mentions)
Cytofix cytoperm kit, by BioLegend (1 mentions)
Facscanto 1, by BD (1 mentions)
3 protocols using cytofix cytoperm kit
1
ILC2 Signaling Profiling and Cytokine Analysis
2
Intracellular Cytokine Staining for Virus-Specific CD8+ T Cells
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ICS was performed using the Cytofix/cytoperm kit (BD Pharmingen, San Diego, CA, USA) according to the manufacturer’s instructions. A total of 1 × 106 freshly isolated splenocytes were stimulated with 1 μM of an individual peptide or inactivated virus in the presence of 1 μg/mL brefeldin A (Sigma-Aldrich, Dorset, UK) in 1000 μL of culture medium at 37 °C for five hours [12 (link)]. Cultures were stimulated with UV-inactivated RSV Long strain, F, NP, and M2-1 peptides as shown in Table 1 . F peptides are recognition sites of palivizumab (synagis®). NP306–314 is based on the human experiments and the others are on mouse. After the stimulation, splenocytes were washed and incubated with an anti-CD8 antibody (R&D Systems, Minneapolis, MN, USA) at 4 °C for 30 min. Splenocytes were fixed with fixation buffer in the Cytofix/cytoperm kit, and intracellular cytokines were stained with a goat IgG antibody against IFN-γ (R&D Systems, USA) and anti-goat IgG PE-Cy7 (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at 4 °C for 60 min. Cellular populations were analyzed using flow cytometry with Cytomics FC 500 (Beckman Coulter, Inc., Indianapolis, IN, USA) and counted until 100,000 cells.
3
Phenotyping and Functional Characterization of Macrophages
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The surface staining for APCCy7-CD11b, APC-CD64, PercP/Cy5.5-CD163, AF488-CD206 and PECy7-CD14 (phenotypic characterization of macrophages) was performed following standard protocol. Briefly, the harvested cells were washed with PBS and blocked in PBS/2% FBS on ice for 30 min. The cells were washed with PBS and the respective Abs cocktails (prepared in PBS/2% FBS) were added to the cell pellet and incubated for 30 min on ice. A fixable viability dye was used according to the manufacturer's instructions to gate on live cells. After washing, the cells were fixed with a Cytofix/Cytoperm Kit (BD Biosciences), washed again and analyzed in a FACS Canto I (Becton Dickinson). All analysis was carried out with FlowJo software (Tree Star). AXL and MERTK expression were evaluated after fixation and permeabilization (Cytofix/Cytoperm Kit) using biotin-conjugated goat anti-human AXL and APC anti-human MERTK mAb IgG1 from R&D. PE-Streptoavidin (Biolegend) was used as detection signal for AXL.
For intracellular staining of TNF-a in monocytes, cells were re-stimulated with ionomycin (1 mg/ml) plus PMA (50 nM) and GolgiStop for an additional 4 h. The cells were then harvested, fixed, permeabilised (Cytofix/Cytoperm Kit) and stained with a PE-conjugated antibody (Ab) against TNF-a. PE Mouse IgG1 k Isotype Ctrl clone MOPC-21 were used.
For intracellular staining of TNF-a in monocytes, cells were re-stimulated with ionomycin (1 mg/ml) plus PMA (50 nM) and GolgiStop for an additional 4 h. The cells were then harvested, fixed, permeabilised (Cytofix/Cytoperm Kit) and stained with a PE-conjugated antibody (Ab) against TNF-a. PE Mouse IgG1 k Isotype Ctrl clone MOPC-21 were used.
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