Tregs were determined by coexpression of CD4, CD25 and Foxp3. After 3 days co-culture with PBMCs and MoDCs [34 (link)], all cells were harvested and stained with anti-porcine CD25 (AbD Serotec), followed by APC goat anti-mouse IgG (H+L) (Life Technologies) and goat anti-porcine CD4a-FITC (Clone 74-12-4; SouthernBiotech). Foxp3 intracellular staining was performed with anti-rat/mouse Foxp3-PE (Clone FJK-16s; eBioscience), which had cross-reactivity with swine Foxp3 [26 (link)], using the Foxp3 Staining Buffer Set (Staining, Fixation/Permeabilization and Permeabilization Buffers; eBioscience), to obtain the three-color staining CD4FITC CD25APC Foxp3PE. The Tregs frequency was evaluated by flow cytometry (BD FACS Canto II), and data were analyzed using FlowJo version 7.6.1 software.
Anti porcine cd25
Manufactured by Bio-Rad
Sourced in United States
Anti-porcine CD25 is a laboratory reagent used for the detection and analysis of CD25, a cell surface marker expressed on activated T cells and regulatory T cells in porcine (pig) samples. This reagent can be used in flow cytometry and other immunoassay applications to identify and study these cell populations.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2, by BD (2 mentions)
Anti rat mouse foxp3 clone fjk 16s, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti mouse immunoglobulin g igg h l, by Thermo Fisher Scientific (1 mentions)
Appropriate isotype controls, by BD (1 mentions)
Fcs express version 5, by De Novo Software (1 mentions)
3 protocols using anti porcine cd25
1
Identification of Porcine Regulatory T Cells
2
Multicolor Flow Cytometry for Porcine T-Cell Analysis
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Flow cytometry was conducted to analyze the frequency of CD4+ cells, CD8+ cells,
interferon γ (IFN-γ), and the ratio of CD4+/CD8+. Cells were stained with 10 µL
of anti-porcine CD25 (AbD Serotec, MCA1736 clone K231.3B2), followed by 0.06 µg
of Alexa Fluor 488 goat anti-mouse immunoglobulin G (IgG, H+L; Invitrogen,
Molecular Probes, Carlsbad, CA, USA). The LYNX rapid antibody conjugation kit
(AbD Serotec) was used to conjugate mouse anti-porcine CD4 with allophycocyanin
(APC; clone 74-12-4; VMRD, Inc., Pullman, Wa, USA) and CD8 with RPECy7 (clone
76-2-11; VMRD, Inc.), and 0.05 µg of these antibodies were then added to the
cell preparation. Forkhead box P3 (Foxp3) intracellular staining was performed
with 0.03 µg of anti-rat/mouse Foxp3 (clone FJK-16s, with cross-reactivity with
swine Foxp3; eBioscience, San Diego, CA, USA) and 0.03 µg of PE-conjugated rat
IgG2a isotype control (clone eBR2a; eBioscience) using the Foxp3 Staining Buffer
Set (Staining, Fixation/Permeabilization, and Permeabilization Buffers;
eBioscience) to obtain the four-color stain
CD4APCCD8RPECy7CD25Alexa488Foxp3PE.
The frequency of regulatory T cells (Tregs) was evaluated by flow cytometry (BD
FACS Canto II), and data were analyzed using BD FACS Diva 6.0 software.
interferon γ (IFN-γ), and the ratio of CD4+/CD8+. Cells were stained with 10 µL
of anti-porcine CD25 (AbD Serotec, MCA1736 clone K231.3B2), followed by 0.06 µg
of Alexa Fluor 488 goat anti-mouse immunoglobulin G (IgG, H+L; Invitrogen,
Molecular Probes, Carlsbad, CA, USA). The LYNX rapid antibody conjugation kit
(AbD Serotec) was used to conjugate mouse anti-porcine CD4 with allophycocyanin
(APC; clone 74-12-4; VMRD, Inc., Pullman, Wa, USA) and CD8 with RPECy7 (clone
76-2-11; VMRD, Inc.), and 0.05 µg of these antibodies were then added to the
cell preparation. Forkhead box P3 (Foxp3) intracellular staining was performed
with 0.03 µg of anti-rat/mouse Foxp3 (clone FJK-16s, with cross-reactivity with
swine Foxp3; eBioscience, San Diego, CA, USA) and 0.03 µg of PE-conjugated rat
IgG2a isotype control (clone eBR2a; eBioscience) using the Foxp3 Staining Buffer
Set (Staining, Fixation/Permeabilization, and Permeabilization Buffers;
eBioscience) to obtain the four-color stain
CD4APCCD8RPECy7CD25Alexa488Foxp3PE.
The frequency of regulatory T cells (Tregs) was evaluated by flow cytometry (BD
FACS Canto II), and data were analyzed using BD FACS Diva 6.0 software.
3
Porcine PBMC and Monocyte Activation Assay
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For PBMC, cells were harvested and stained with anti-porcine γδ TCR (Clone PGBL22A; Monoclonal Antibody Centre, Washington State University, Pullman, USA) and anti-porcine CD25 (Clone K231.3B2; AbD serotec, Kidlington, UK). CD25 (IL-2Rα ) was used as a common and well-studied marker of peripheral T-cell activation (Caruso et al., 1997; Jouen-Beades et al., 1997) . The mAbs were conjugated to either FITC or APC fluorophores using Zenon antibody labelling kits (Invitrogen), according to the manufacturer's instructions. For monocytes, cells were stained for CD80/86 expression using human CTLA-4/mouse IgG-FITC fusion protein (Ancell, Bayport, USA). Appropriate isotype controls (BD biosciences) were included in every experiment. Cells were acquired on an Accuri C6 flow cytometer. Gating for lymphocytes or monocytes was carried out based on forward/side scatter and at least 10000 events were acquired. Data were analysed using FCS express version 5 (De Novo Software, Glendale, CA).
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