Peripheral CD4+ T cells were enriched with the EasySep Human CD4+T cells kit (STEMCELL Technologies) from the blood of patients, heterozygous relatives, and control donors. Enriched CD4+ T cells were stained with the following antibodies: APC-Cy7-anti-CD4, PE-anti-CD25, PerCP-Cy5.5-anti-CD127, AlexaFluor700-anti-HLA-DR (all from BioLegend) and with AlexaFluor647-anti-Helios (BioLegend), Alexa Fluor 488-anti-FOXP3, and eV605-anti-CD3 (both from eBioscience) after fixation and permeabilization of T cells (gating strategy is shown in figs. S23 and S24 ). After staining enriched CD4+ T cells with APC-Cy7-anti-CD4, PE-anti-CD25, and Alexa Fluor 647-anti-CD127 (BioLegend), CD4+CD127+Tconv cells and CD4+CD25hiCD127−/lo Treg cells from patients, heterozygous relatives, and HDs were sorted using a FACSAria flow cytometer (Becton Dickinson, Mountain View, CA). CD4+CD25+CD127+ Tconv cells were labeled with CellTrace carboxyfluorescein diacetate succinimidyl ester (CFSE) (InVivogen). Cocultures of Treg and Tconv at 1:1, 1:4, 1:8, and 1:16 ratios were stimulated with the Treg Suppression Inspector Human kit (Miltenyi Biotec) at a 1 bead/1 cell ratio. Proliferation of the viable Tconv was analyzed by CFSE dilution at day 3.5.
Anti cd25 pe
Manufactured by BioLegend
Sourced in United States
Anti-CD25-PE is a fluorescently-labeled antibody that binds to the CD25 antigen, which is expressed on the surface of activated T cells and regulatory T cells. This product can be used for flow cytometry applications to identify and analyze these cell populations.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd4 fitc, by BioLegend (9 mentions)
Anti cd3 fitc, by BioLegend (4 mentions)
Anti cd4 apc, by BioLegend (4 mentions)
Anti foxp3 apc, by Thermo Fisher Scientific (4 mentions)
Anti cd4 apc cy7, by BioLegend (3 mentions)
Anti ifn γ pe, by BioLegend (3 mentions)
Anti cd8 apc, by BioLegend (3 mentions)
Anti cd8 pe cy7, by BD (3 mentions)
Anti human ifn γ fitc, by BioLegend (2 mentions)
Anti human il 10 apc, by BioLegend (2 mentions)
Phorbol myristate acetate (pma), by Merck Group (2 mentions)
Ionomycin, by Merck Group (2 mentions)
Transcription factor buffer set, by BD (2 mentions)
Alexa fluor 647 anti foxp3, by BD (2 mentions)
Percp cy5.5 anti cd4, by BioLegend (2 mentions)
Anti il 17 pe, by BioLegend (2 mentions)
Anti cd62l fitc, by BioLegend (2 mentions)
Anti igm fitc, by BioLegend (2 mentions)
Anti foxp3 fitc, by BioLegend (2 mentions)
Anti ly6c pe, by BioLegend (2 mentions)
36 protocols using anti cd25 pe
1
Suppressive Function of Regulatory T Cells
2
Isolation and Co-culture of Murine T and B Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Splenic CD4+ CD25− T cells and B220+ B cells were isolated from BALB/c mice. Splenic B220+ B cells were immunomagnetically purified using a BD IMag Cell Separation Magnet. Splenic CD4+ CD25− T cells were purified by negative immunomagnetic selection using the EasySep Mouse CD4+ T Cell Isolation Kit (STEMCELL Technologies). CD4+CD25+ tTreg cells were obtained by incubating PE‐anti‐CD25 (BioLegend) and anti‐PE beads (BD Biolegend). Purified B cells were co‐cultured with purified splenic CD4+ CD25− T cells under anti‐CD3/CD28 (0.5 μg/ml) stimulation for 3 days. Treg‐of‐B cells were obtained by depleting B220+ B cells.
3
Phenotyping T Cells Co-cultured with MenSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Flow cytometry experiments were performed to assess T cells co-cultured with MenSCs. The fluorochrome-conjugated antibodies included Alexa Fluor 647 anti-FoxP3 (BD Biosciences, USA), FITC anti-human IFN-γ, PerCP/Cy5.5 anti-CD4, APC anti-human IL-10, and PE anti-CD25 (All from Biolegend, USA) were employed. Antibodies were used at the concentrations recommended by the manufacturers. For assessment of cytokines, cells were treated with 50 µg/mL PMA (Sigma, USA), 1 µg/mL ionomycin (Sigma, USA) and 0.7 µg/mL Monensin (BD Biosciences, USA) 6 h. before staining procedure. For intracellular staining of Foxp3 and cytokines, transcription factor buffer set (BD Biosciences, USA) was used for cell permeabilization and fixation according to the manufacturer’s instruction. Cells were also stained with Live/Dead fixable near red fluorescent dye (Molecular Probes, USA) to separate the alive and dead populations before antibody-specific gating. The stained cell was then read by flow cytometer Attune NxT flow cytometer and analyzed by FlowJo software (FlowJo, LLC, USA).
4
Isolation and Characterization of CD4+ T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Peripheral blood mononuclear cells (PBMC) were isolated as described previously [25 (link)]. For the PBMC preparations, CD4+ T cells was isolated and purified by automated magnetic sorting and anti-CD4 microbeads isolation Kits (Miltenyi Biotec) according to the manufacturer’s protocol. All sorted cell populations exhibited 95% purity, as revealed by flow cytometry (Additional file 1 ).
The OVA-primed CD4+ T cells were, respectively incubated with OX40L-Ig fusion protein or control IgG for 48 h. To estimate proliferation of T cells, 3H-TdR was added during the last 12 h of a 48 h culture [26 (link)]. Cells were harvested and counted with liquid scintillation counting (Aloka Lsc-lb7, shanghai, china). IL-4, IFN-γ, IL-17 and TGF-βlevels in supernatants were measured by ELISA according to the manufacturer’s protocol.
Flow cytometric analysis of Th1, Th2 and Th17 was performed using FITC anti-CD4 (Biolegend, San Diego, CA, USA), PE anti-IFN-γ (Biolegend), PE anti- IL-4 (Biolegend), PE anti-IL-17 (Biolegend). For analysis of Treg cells, the cells were stained with FITC anti-CD4 and PE anti-CD25 (Biolegend) and then incubated with PE anti-Foxp3 (eBioscience, San Diego, CA, USA).
The OVA-primed CD4+ T cells were, respectively incubated with OX40L-Ig fusion protein or control IgG for 48 h. To estimate proliferation of T cells, 3H-TdR was added during the last 12 h of a 48 h culture [26 (link)]. Cells were harvested and counted with liquid scintillation counting (Aloka Lsc-lb7, shanghai, china). IL-4, IFN-γ, IL-17 and TGF-βlevels in supernatants were measured by ELISA according to the manufacturer’s protocol.
Flow cytometric analysis of Th1, Th2 and Th17 was performed using FITC anti-CD4 (Biolegend, San Diego, CA, USA), PE anti-IFN-γ (Biolegend), PE anti- IL-4 (Biolegend), PE anti-IL-17 (Biolegend). For analysis of Treg cells, the cells were stained with FITC anti-CD4 and PE anti-CD25 (Biolegend) and then incubated with PE anti-Foxp3 (eBioscience, San Diego, CA, USA).
5
Multiparametric Flow Cytometry Panel
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For flow cytometry, we used FITC anti-CD4, APC anti-CD8, FITC anti-IgM, APC anti-CXCR5, PE anti-PD1, FITC anti-CD62L, PE anti-CD25, APC anti-LAG-3, APC anti-TIM-3, PE anti-PD-1 and FITC anti-ICOS, FITC anti-Annexin V and APC anti-FoxP3 antibodies (all from BioLegend).
6
Splenocyte Isolation and Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The spleens were aseptically removed and disrupted by mechanical dissociation. After filtration through a 100-μm nylon screen, splenocytes were collected, washed, and suspended with red blood cell lysing buffer. The splenocytes were suspended in diluted staining perm wash buffer (BioLegend, San Diego, CA) at a concentration of 1 × 107 cells/ml. Cell suspensions or blood samples 100 μl were stained with FITC-anti-CD3 (1:500, BioLegend, San Diego, CA, USA), APC-anti-CD4 (1:100, BioLegend), PerCP-anti-CD8a (1:100, BioLegend), PE-anti-CD25 (1:100, BioLegend) and APC-Cy7 anti-CD45 (1:100, BioLegend) or isotype-matched controls (1:200, BioLegend), according to the manufacturer’s instructions. The samples were mixed gently, incubated for 20 min at 4 °C in the darkness and subsequently analyzed using FACS Verse flow (Becton Dickinson, CA, USA). The gating strategies are shown in Additional file 1 .
7
In Vitro Generation of OVA-Specific Memory CD8+ T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In Vitro Memory Differentiation Memory OT-I T cells were generated as described previously (Balmer et al., 2016; van der Windt et al., 2013) . Briefly, the lymph nodes from MHC class I-restricted OVA-specific T cell receptor (OT-I) transgenic mice and the spleen of C57BL/6 mice were aseptically removed and incubated in liberase TL (Roche) for 30 min. After mashing through a 70 mm cell strainer (BD Biosciences), red blood cells were lysed with RBC Lysis Buffer Solution (eBioscience). The isolated cell suspensions were washed in RPMI medium (RPMI 1640 containing 10% FCS, 100 U/mL penicillin, 100 mg streptomycin, 0.29 mg/mL L-glutamine, 50 mM 2-Mercaptoethanol (Life Technologies)) and re-suspended to 10 6 cells/mL. The splenocytes and OT-I cells were pooled in a ratio of 1:1 and activated with OVA peptide (Eurogentec) at 10 À9 M at 37 C for 3 days. The cells were then washed and re-suspended to 2 3 10 6 cells/mL and cultured in the presence of IL-15 (10 ng/mL) at 37 C for another 3 days to generate OVA-specific memory CD8 + T cells. Phenotyping was performed using BUV395-anti CD44 (BD), APC-anti CD8, BV421-anti KLRG1, PE-anti-CD62L, APC-Cy7-anti CD43, BV421-anti PDL1, PE-anti-CD25, BV510-anti CD27 (all Biolegend) and PE-Cy5-anti CD127 (eBioscience).
8
Treg Cell Activation in Mice
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Spleens from 5 mice of each experimental group were harvested on days 0, 2, 4, 6, and 8 p.i. The spleen of day 0 was labeled as control. To measure the activation of regulatory T cells (Tregs), 107 fresh splenocytes were stained with FITC-anti-CD4, and PE-anti-CD25 for surface staining in cell staining buffer (Biolegend, SanDiego, California, USA). After the cells were fixed and permeabilized, intracytoplasmic staining by labeling with fluorochrome conjugated antibody of APC-anti-Foxp3 was performed. After that, the resuspended, fixed and intracellularly labeled cells were analyzed on FACSCalibur flow cytometers (BD Bioscience, San Jose, California, USA). Data were analyzed with FlowJo software (Treestar). The experiments were replicated 3 times.
9
Immunophenotyping of T Cell Subsets
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The obtained blood samples were separated to plasma and lymphocytes. Lymphocytes were stained (surface and intracellular) to determine Treg+ cells CD4+CD25+FOXP3+ and Th1 CD3+CD4+CD25− population in treated groups in comparison to PBS. First, cells were stained by Percp-Anti-CD3, FITC-Anti-CD4, and PE-Anti-CD25 (Biolegend, USA) for 30 min on ice. Then it was fixed, permeabilized and stained by Alexa Fluor 647- anti- Foxp3 (Biolegend, USA) for 30 min on ice. Finally cells were examined by BD Accuri C6. Later, plasma samples were utilized to determine periphery TGF-β and IL-12 cytokines by ELISA kits according to manufacturer instructions (Lington Biosciences, China).
10
Immunophenotyping of T cells co-cultured with MenSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Flow cytometry experiments were performed to assess T cells co-cultured with MenSCs. The uorochrome-conjugated antibodies included Alexa Fluor 647 anti FoxP3 (BD Bioscience, USA), FITC antihuman IFN-γ, PerCP/Cy5.5 anti-CD4, APC anti-human IL-10, and PE anti-CD25 (All from Biolegend, USA).
Antibodies were used at the concentrations recommended by the manufacturers. For assessment of cytokines, cells were treated with 50 µg/ml PMA (Sigma, USA), 1 µg/ml ionomycin (Sigma, USA) and 0.
Antibodies were used at the concentrations recommended by the manufacturers. For assessment of cytokines, cells were treated with 50 µg/ml PMA (Sigma, USA), 1 µg/ml ionomycin (Sigma, USA) and 0.
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!