Mice were sacrificed and their spleens were harvested. The spleen samples were dissociated into single-cell suspensions and 2×106 cells were transferred to round-bottom polystyrene tubes (BD Biosciences, CA, USA). After washing with PBS supplemented with 2% FBS, mouse spleen cells were stained with a rat anti-mouse CD4 (2 μg/mL) antibody conjugated with allophycocyanin (APC) (eBioscience, CA, USA) and a rat anti-mouse CD25 (2 μg/mL) antibody conjugated with APC-Cy7 (BD Pharmingen, CA, USA). The cells were then permeabilized using Flow Cytometry Fixation and Permeabilization buffer (eBioscience), and stained with an anti-human/mouse RORγt (2 μg/mL) antibody conjugated with phycoerythrin (PE) (eBioscience) and an anti-mouse FoxP3 (5 μg/mL) antibody conjugated with fluorescein isothiocyanate (FITC) (eBioscience). Flow cytometry analysis was performed using a BD LSR Fortessa cell analyzer (BD Biosciences). To analyze the data, FlowJo V10 Single Cell Analysis Software was used (TreeStar Inc., OR, USA).
Phycoerythrin pe
Manufactured by Thermo Fisher Scientific
Sourced in United States
Phycoerythrin (PE) is a fluorescent protein derived from red algae. It is used as a fluorescent label in various biological applications, such as flow cytometry and immunoassays, due to its ability to emit bright red-orange fluorescence when excited by a suitable light source.
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Lab products found in correlation
Fluorescein isothiocyanate (fitc), by Thermo Fisher Scientific (2 mentions)
Lsrfortessa cell analyzer, by BD (2 mentions)
Polystyrene round bottom tube, by BD (1 mentions)
Flow cytometry fixation and permeabilization buffer, by Thermo Fisher Scientific (1 mentions)
Apc cy7, by BD (1 mentions)
Apc conjugated rat anti mouse cd4 antibody, by BD (1 mentions)
Foxp3 staining buffer set, by Thermo Fisher Scientific (1 mentions)
Fluorescein isothiocyanate (fitc), by Enzo Life Sciences (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Facscalibur, by BD (1 mentions)
Cellquest software, by BD (1 mentions)
Facsverse, by BD (1 mentions)
5 protocols using phycoerythrin pe
1
Flow Cytometric Analysis of Mouse Splenocytes
2
Detecting Murine Th17 Cells by Flow Cytometry
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To detect Th17 cells in the mouse spleen, splenocytes were isolated from each group. The cells were incubated with allophycocyanin (APC)-conjugated rat anti-mouse CD4 antibody (catalogue number, 561091; BD Biosciences, San Jose, CA, USA), and the cells were permeabilized using a Foxp3 staining buffer set (00-5523; eBioscience, San Diego, CA, USA). The Th17-specific marker RORγt was detected by anti-human/mouse RORγt antibody conjugated with phycoerythrin (PE) (eBioscience, 12-6988). After the overall cellular staining process, the cell populations were identified and estimated using an LSRFortessa cell analyzer (BD Biosciences). The acquired data were analyzed using FlowJo version 7.6.5 software (TreeStar Inc. Ashland, OR, USA). Cells were first gated for CD4+ proportion and analyzed for the expression of RORγt.
3
Regulatory T Cell Phenotyping in Lamina Propria
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The following monoclonal antibodies, that is, anti-rat cluster of differentiation 4 (CD4) antibody (mouse IgG, Catlog: 11-0010-82, eBioscience, Basingstoke, UK), anti-rat cluster of differentiation 25 (CD25) antibody (mouse IgG, Catlog: 12-0390-82, eBioscience), and anti-rat Foxp3 antibody (rat IgG, Catlog: 56-5773-82, eBioscience), were conjugated with fluorescein isothiocyanate (FITC) allophycocyanin (APC), and phycoerythrin (PE) purchased from eBioscience. Lamina propria cells were washed with HBSS, and the isolated cells were suspended in the respective tube with 100 μL HBSS. Subsequently, the cells were stained with monoclonal antibodies, that is, anti-rat CD4 (mouse IgG, Catlog: 11-0010-82, eBioscience), anti-rat CD25 (mouse IgG, Catlog: 12-0390-82, eBioscience), and anti-rat Foxp3 (rat IgG, Catlog: 56-5773-82, eBioscience). For the intracellular staining of Foxp3, the cells were intubated with Fc gamma receptor (FcγR)-blocking monoclonal antibody before the staining for surface antigens under the manufacturer’s instructions.
4
Flow Cytometric Analysis of DC Activation
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DCs were treated with 50 µg/ml of the ethanol-killed S. aureus strain for 48 h, or with Pam3CSK4 at 0–10 μg/ml for 0 to 72 h. Following stimulation, the cells were stained with anti-mouse antibodies conjugated with fluorescent dyes as previously described (33 (link)). For membrane-bound BAFF (mBAFF) analysis, DCs were stained with anti-mouse BAFF antibodies conjugated with fluorescein isothiocyanate (FITC) (Enzo Life Science, Plymouth Meeting, PA, USA) together with anti-mouse CD11c antibodies conjugated with allophycocyanin (APC) (BD Biosciences). Additionally, DCs were stained with antibodies for mouse TLR1 conjugated with Alexa Fluor 647 or TLR2 conjugated with phycoerythrin (PE) (e-Bioscience, San Diego, CA, USA). The cells were then subjected to flow cytometry (FACSCalibur) with CellQuest software (BD Biosciences). Data were analyzed using FlowJo software (Tree Star, San Carlos, CA, USA).
5
Multicolor Flow Cytometry Staining
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Anti-mouse VISTA (MIH63, rat IgG2a mAb) was generated as previously described. [19] [20] [21] For fluorescence immunohistochemistry or flow cytometry, mAbs against CD3 (145-2C11, Armenian hamster IgG), CD4 (GK1.5, rat IgG2b), CD8 (53.6.72, rat IgG2a), CD11b (M1/70, rat IgG2b), CD25 (PC61.5, rat IgG1), Foxp3 (FJK-16s, rat IgG2a), CD103 (M290, rat IgG2a), and anti-VISTA mAb were used. All fluorescein isothiocyanate (FITC)-, Phycoerythrin (PE)-, allophycocyanin (ACP)-, and biotin-conjugated mAbs and isotype control Igs were obtained from eBioscience (San Diego, CA, USA). Culture supernatant from the 2.4G2 hybridoma (anti-CD16/CD32 mAb) was used to block non-specific binding. Stained cells were then analyzed (BD FACSverse, BD Biosciences, San Jose, CA, USA; Flow Jo software, Tomy Digital Biology, Tokyo, Japan).
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