PD-L1 regulates the development, maintenance, and function of induced regulatory T cells methods
Show snapshot details
On this page
This experiment, in seven questions
Jump straight to the part of the recipe you need. Data and provenance labels stay close to the action they support.
Shopping and prep list
What do I need before I start?
Biological model pending
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Before you run
What should be confirmed before execution?
First confirmation
Species or subject information is missing.
Confirm before execution
Protocol does not list structured equipment.
Confirm before execution
This page shows passages quoted from the paper. Equipment, materials, times, and temperatures are hidden until the page is rebuilt from the full paper and checked by a reviewer. Check the paper before using this method.
Confirm before execution
Open the source paper before finalizing run-specific details.
Procurement checkpoint
Use source-stated vendors where present. Treat mapped products as sourcing options unless the page marks an exact source match.
Open quote workflowStep-by-step procedure
What do I do, in order?
Reagents.
The following anti-mouse antibodies were used in cell surface staining, intracellular cytokine staining, and epoxy bead conjugation: anti-CD16/CD32 (Fc Block), CD4 PerCP-Cy5.5 (clone RM4-5), CD62L PE (clone MEL-14), IL-2 APC (clone JES6-5H4; eBioscience), CD45.1 APC (clone A20), IL-17 PE (clone TC11-18H10), and IFN-γ PE (clone XMG1.2; BD). Anti-CD3 (clone 2C11) plus anti-CD28 (clone 37.51) were used for bead conjugation and were purchased from Bio X Cell. Cells were sorted on a FACSAria cell sorter (BD). Cell surface staining was performed at 4°C in FACS Buffer (1% FCS, PBS, 2 mM EDTA; Invitrogen). CFSE was purchased from Invitrogen.
In vitro iT reg cell development.
Anti-CD3 (clone 2C11; Bio X Cell) plus anti-CD28 (clone 37.51; Bio X Cell) were covalently attached to Dynabeads M450 glycidyl ether beads according to the manufacturer's directions (Invitrogen). We ensured equal loading of proteins during preparation by keeping constant the total amount of protein (antibodies and fusion proteins) at 5 µg per 10 7 beads as previously described (; ). In general, 10 7 beads were coated with 1 µg of anti-CD3 (20% of total protein), 1 µg of anti-CD28, and either 60% control human IgG1 (referred to control Ig beads; Bio X Cell) or 40% PD-L1-hIgG1 Fc fusion protein (referred to as PD-L1-Ig beads 40; R& D Systems) plus 20% control human IgG1 Fc. In some experiments, increasing amounts of PD-L1-Ig was used to coat the epoxy beads (20, 40, and 60% of total protein per 10 7 beads = 1, 2, and 3 µg of PD-L1-Ig per 10 7 beads...
In vitro iT reg cell development.
CD4 + CD62L + Foxp3 - naive T cells were cultured with beads at a fixed ratio of 1:5 (T cells/beads). In brief, 1-2 × 10 6 T cells were plated at 10 6 /ml in a 24-well flat-bottom tissue culture plate with beads in complete media consisting of RPMI-1640 with L-glutamine (Invitrogen) supplemented with 10% FCS (Sigma-Aldrich), penicillin-streptomycin (100 U penicillin and 100 µg streptomycin; Invitrogen), 12 mM HEPES (Invitrogen), and 50 µM β-mercaptoethanol (Sigma-Aldrich) plus 2 ng/ml TGF-β (R&D Systems) and 20 U/ml rh-IL2 (R&D Systems) for 3 d at 37°C with 5% CO 2. In some experiments, sorted CD4 + CD25 - CD62L + CD44 low naive T cells were labeled with 1 µM CFSE before culture with beads and TGF-β for 3 d at 37°C with 5% CO 2. T cells were then stained for Foxp3 expression (eBioscience). To further evaluate the effect o...
In vitro suppression assays.
For CFSE dilution experiments, CD4 + CD25 - CD45.1 + naive T eff cells were labeled with 1 mM CFSE for 10 min in RPMI-1640 (serum free) and washed twice with 100% FBS and twice with complete media before culture. 10 5 T eff cells were cultured with 10 5 iT reg cells and PD-L1-Ig beads (5:1 bead/T eff cell ratio) in 96-well flat-bottom plates (BD). 72 h later, CD4 + CD45.1 + T cells were gated and analyzed for CFSE dilution. Division index (defined as the mean number of divisions that a cell has undergone) was calculated using FlowJo Proliferation analysis software (Tree Star, Inc.). For CFSE dilution experiments interrogating the suppressive capacity of PD-L1-iT reg cells versus control iT reg cells, naive T cells were induced toward T reg cell development in vitro using PD-L1 or control beads in the presence of TGF-β and IL-2 for 3 d, as described in the previous sec...
Phospho-flow cytometry.
Naive CD4 + T cells were sorted from 2D2 Foxp3.GFP reporter mice and cultured with either PD-L1-Ig beads or control beads in the presence of 2 ng/ml TGF-β and 20 U/ml IL-2 for 18 h. Signaling molecules were assessed with antibodies against phospho-Akt Ser473 Alexa Fluor 647 (clone D9E), phospho-mTOR Ser24448 (clone 49F9), phospho-S6 Ser235/236 Alexa Fluor 647 (clone D57.2.2E), and PTEN Alexa Fluor 647 (clone 138G6; Cell Signaling Technology). Isotype control staining was performed using rabbit IgG isotype mAb Alexa Fluor 647 (DA1E; Cell Signaling Technology). p-mTOR was detected with anti-rabbit Alexa Fluor 647 secondary (Invitrogen). Intracellular staining was performed as described in the manufacturer's protocol. In brief, T cells were collected and washed thoroughly with PBS in 96-well V-bottom plates. Cells were then fixed with 2% paraformaldehyde for 10 min at 37...
Measurement outputs
What raw and processed outputs should exist?
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
Review raw outputs for quality, remove unusable captures, and organize the data into a comparison-ready table or image set.
inferred from protocolScoring or quantification
Quantify the primary endpoint described in the methods before moving into any statistical comparison.
inferred from protocolStatistical comparison
Statistical method not yet structured for this page.
inferred from protocolReporting output
Report representative outputs alongside the summary comparison used to support the conclusion.
inferred from protocolSource and audit
What supports the facts on this page?
Evidence quotes (5)
The following anti-mouse antibodies were used in cell surface staining, intracellular cytokine staining, and epoxy bead conjugation: anti-CD16/CD32 (Fc Block), CD4 PerCP-Cy5.5 (clone RM4-5), CD62L PE (clone MEL-14), IL-2 APC (clone JES6-5H4; eBioscience), CD45.1 APC (clone A20), IL-17 PE (clone TC11-18H10), and IFN-γ PE (clone XMG1.2; BD). Anti-CD3 (clone 2C11) plus anti-CD28 (clone 37.51) were used for bead conjugation and were purchased from Bio X Cell. Cells were sorted on a FACSAria cell sorter (BD). Cell surface staining was performed at 4°C in FACS Buffer (1% FCS, PBS, 2 mM EDTA; Invitrogen). CFSE was purchased from Invitrogen.
Anti-CD3 (clone 2C11; Bio X Cell) plus anti-CD28 (clone 37.51; Bio X Cell) were covalently attached to Dynabeads M450 glycidyl ether beads according to the manufacturer's directions (Invitrogen). We ensured equal loading of proteins during preparation by keeping constant the total amount of protein (antibodies and fusion proteins) at 5 µg per 10 7 beads as previously described (; ). In general, 10 7 beads were coated with 1 µg of anti-CD3 (20% of total protein), 1 µg of anti-CD28, and either 60% control human IgG1 (referred to control Ig beads; Bio X Cell) or 40% PD-L1-hIgG1 Fc fusion protein (referred to as PD-L1-Ig beads 40; R& D Systems) plus 20% control human IgG1 Fc. In some experiments, increasing amounts of PD-L1-Ig was used to coat the epoxy beads (20, 40, and 60% of total protein per 10 7 beads = 1, 2, and 3 µg of PD-L1-Ig per 10 7 beads). In these cases, the remaining surface of the beads were coated with control human IgG1. Covalent attachment of the proteins to the beads was performed in NaPO 4 buffer for 24 h at room temperature on a Nutating Mixer (Lab-Tech Incorporated). Beads were then washed three times in PBS over a magnet...
CD4 + CD62L + Foxp3 - naive T cells were cultured with beads at a fixed ratio of 1:5 (T cells/beads). In brief, 1-2 × 10 6 T cells were plated at 10 6 /ml in a 24-well flat-bottom tissue culture plate with beads in complete media consisting of RPMI-1640 with L-glutamine (Invitrogen) supplemented with 10% FCS (Sigma-Aldrich), penicillin-streptomycin (100 U penicillin and 100 µg streptomycin; Invitrogen), 12 mM HEPES (Invitrogen), and 50 µM β-mercaptoethanol (Sigma-Aldrich) plus 2 ng/ml TGF-β (R&D Systems) and 20 U/ml rh-IL2 (R&D Systems) for 3 d at 37°C with 5% CO 2. In some experiments, sorted CD4 + CD25 - CD62L + CD44 low naive T cells were labeled with 1 µM CFSE before culture with beads and TGF-β for 3 d at 37°C with 5% CO 2. T cells were then stained for Foxp3 expression (eBioscience). To further evaluate the effect of IL-2 on PD-L1-mediated T reg cell development, some experiments were conducted in the absence of exogenous rh-IL2 (as noted in the legend). To analyze the effect of TGF-β on PD-L1-mediated T reg cell development, we performed some experiments using a range of TGF-β concentrat...
For CFSE dilution experiments, CD4 + CD25 - CD45.1 + naive T eff cells were labeled with 1 mM CFSE for 10 min in RPMI-1640 (serum free) and washed twice with 100% FBS and twice with complete media before culture. 10 5 T eff cells were cultured with 10 5 iT reg cells and PD-L1-Ig beads (5:1 bead/T eff cell ratio) in 96-well flat-bottom plates (BD). 72 h later, CD4 + CD45.1 + T cells were gated and analyzed for CFSE dilution. Division index (defined as the mean number of divisions that a cell has undergone) was calculated using FlowJo Proliferation analysis software (Tree Star, Inc.). For CFSE dilution experiments interrogating the suppressive capacity of PD-L1-iT reg cells versus control iT reg cells, naive T cells were induced toward T reg cell development in vitro using PD-L1 or control beads in the presence of TGF-β and IL-2 for 3 d, as described in the previous section, Foxp3.GFP + T cells were sorted on a FACSAria cell sorter. Control and PD-L Foxp3.GFP + iT reg cells were then co-cultured with CFSE-labeled CD4 + CD25 - Thy1.1 + T eff cells and stimulated with control beads (containing anti-CD3, anti-CD28, and control Ig). 10 5 CD4 + CD25 - Thy1....
Naive CD4 + T cells were sorted from 2D2 Foxp3.GFP reporter mice and cultured with either PD-L1-Ig beads or control beads in the presence of 2 ng/ml TGF-β and 20 U/ml IL-2 for 18 h. Signaling molecules were assessed with antibodies against phospho-Akt Ser473 Alexa Fluor 647 (clone D9E), phospho-mTOR Ser24448 (clone 49F9), phospho-S6 Ser235/236 Alexa Fluor 647 (clone D57.2.2E), and PTEN Alexa Fluor 647 (clone 138G6; Cell Signaling Technology). Isotype control staining was performed using rabbit IgG isotype mAb Alexa Fluor 647 (DA1E; Cell Signaling Technology). p-mTOR was detected with anti-rabbit Alexa Fluor 647 secondary (Invitrogen). Intracellular staining was performed as described in the manufacturer's protocol. In brief, T cells were collected and washed thoroughly with PBS in 96-well V-bottom plates. Cells were then fixed with 2% paraformaldehyde for 10 min at 37°C. After fixation, plates were prechilled on ice for 1 min before permeabilization by slowly adding ice-cold methanol to a final concentration of 90% methanol. Cells were then incubated on ice for 30 min for permeabilization before being washed with 1% FCS/PBS (incubation buffer). Cells were b...
Machine-readable layer
[
{
"@context": "https://schema.org",
"@type": "HowTo",
"name": "PD-L1 regulates the development, maintenance, and function of induced regulatory T cells methods",
"description": "Protocol for PD-L1 regulates the development, maintenance, and function of induced regulatory T cells methods",
"step": [
{
"@type": "HowToStep",
"position": 1,
"name": "Reagents.",
"text": "The following anti-mouse antibodies were used in cell surface staining, intracellular cytokine staining, and epoxy bead conjugation: anti-CD16/CD32 (Fc Block), CD4 PerCP-Cy5.5 (clone RM4-5), CD62L PE (clone MEL-14), IL-2 APC (clone JES6-5H4; eBioscience), CD45.1 APC (clone A20), IL-17 PE (clone TC11-18H10), and IFN-γ PE (clone XMG1.2; BD). Anti-CD3 (clone 2C11) plus anti-CD28 (clone 37.51) were used for bead conjugation and were purchased from Bio X Cell. Cells were sorted on a FACSAria cell sorter (BD). Cell surface staining was performed at 4°C in FACS Buffer (1% FCS, PBS, 2 mM EDTA; Invitrogen). CFSE was purchased from Invitrogen."
},
{
"@type": "HowToStep",
"position": 2,
"name": "In vitro iT reg cell development.",
"text": "Anti-CD3 (clone 2C11; Bio X Cell) plus anti-CD28 (clone 37.51; Bio X Cell) were covalently attached to Dynabeads M450 glycidyl ether beads according to the manufacturer's directions (Invitrogen). We ensured equal loading of proteins during preparation by keeping constant the total amount of protein (antibodies and fusion proteins) at 5 µg per 10 7 beads as previously described (; ). In general, 10 7 beads were coated with 1 µg of anti-CD3 (20% of total protein), 1 µg of anti-CD28, and either 60% control human IgG1 (referred to control Ig beads; Bio X Cell) or 40% PD-L1-hIgG1 Fc fusion protein (referred to as PD-L1-Ig beads 40; R& D Systems) plus 20% control human IgG1 Fc. In some experiments, increasing amounts of PD-L1-Ig was used to coat the epoxy beads (20, 40, and 60% of total protein per 10 7 beads = 1, 2, and 3 µg of PD-L1-Ig per 10 7 beads..."
},
{
"@type": "HowToStep",
"position": 3,
"name": "In vitro iT reg cell development.",
"text": "CD4 + CD62L + Foxp3 - naive T cells were cultured with beads at a fixed ratio of 1:5 (T cells/beads). In brief, 1-2 × 10 6 T cells were plated at 10 6 /ml in a 24-well flat-bottom tissue culture plate with beads in complete media consisting of RPMI-1640 with L-glutamine (Invitrogen) supplemented with 10% FCS (Sigma-Aldrich), penicillin-streptomycin (100 U penicillin and 100 µg streptomycin; Invitrogen), 12 mM HEPES (Invitrogen), and 50 µM β-mercaptoethanol (Sigma-Aldrich) plus 2 ng/ml TGF-β (R&D Systems) and 20 U/ml rh-IL2 (R&D Systems) for 3 d at 37°C with 5% CO 2. In some experiments, sorted CD4 + CD25 - CD62L + CD44 low naive T cells were labeled with 1 µM CFSE before culture with beads and TGF-β for 3 d at 37°C with 5% CO 2. T cells were then stained for Foxp3 expression (eBioscience). To further evaluate the effect o..."
},
{
"@type": "HowToStep",
"position": 4,
"name": "In vitro suppression assays.",
"text": "For CFSE dilution experiments, CD4 + CD25 - CD45.1 + naive T eff cells were labeled with 1 mM CFSE for 10 min in RPMI-1640 (serum free) and washed twice with 100% FBS and twice with complete media before culture. 10 5 T eff cells were cultured with 10 5 iT reg cells and PD-L1-Ig beads (5:1 bead/T eff cell ratio) in 96-well flat-bottom plates (BD). 72 h later, CD4 + CD45.1 + T cells were gated and analyzed for CFSE dilution. Division index (defined as the mean number of divisions that a cell has undergone) was calculated using FlowJo Proliferation analysis software (Tree Star, Inc.). For CFSE dilution experiments interrogating the suppressive capacity of PD-L1-iT reg cells versus control iT reg cells, naive T cells were induced toward T reg cell development in vitro using PD-L1 or control beads in the presence of TGF-β and IL-2 for 3 d, as described in the previous sec..."
},
{
"@type": "HowToStep",
"position": 5,
"name": "Phospho-flow cytometry.",
"text": "Naive CD4 + T cells were sorted from 2D2 Foxp3.GFP reporter mice and cultured with either PD-L1-Ig beads or control beads in the presence of 2 ng/ml TGF-β and 20 U/ml IL-2 for 18 h. Signaling molecules were assessed with antibodies against phospho-Akt Ser473 Alexa Fluor 647 (clone D9E), phospho-mTOR Ser24448 (clone 49F9), phospho-S6 Ser235/236 Alexa Fluor 647 (clone D57.2.2E), and PTEN Alexa Fluor 647 (clone 138G6; Cell Signaling Technology). Isotype control staining was performed using rabbit IgG isotype mAb Alexa Fluor 647 (DA1E; Cell Signaling Technology). p-mTOR was detected with anti-rabbit Alexa Fluor 647 secondary (Invitrogen). Intracellular staining was performed as described in the manufacturer's protocol. In brief, T cells were collected and washed thoroughly with PBS in 96-well V-bottom plates. Cells were then fixed with 2% paraformaldehyde for 10 min at 37..."
}
],
"tool": [],
"supply": [],
"isBasedOn": {
"@type": "ScholarlyArticle",
"headline": "PD-L1 regulates the development, maintenance, and function of induced regulatory T cells",
"datePublished": "2009",
"author": [
{
"@type": "Person",
"name": "Loise M. Francisco"
},
{
"@type": "Person",
"name": "Victor H. Salinas"
},
{
"@type": "Person",
"name": "Keturah E. Brown"
},
{
"@type": "Person",
"name": "Vijay K. Vanguri"
},
{
"@type": "Person",
"name": "Gordon J. Freeman"
},
{
"@type": "Person",
"name": "Vijay K. Kuchroo"
},
{
"@type": "Person",
"name": "Arlene H. Sharpe"
}
],
"identifier": "10.1084/jem.20090847"
}
},
{
"@context": "https://schema.org",
"@type": "BreadcrumbList",
"itemListElement": [
{
"@type": "ListItem",
"position": 1,
"name": "Experiments",
"item": "https://replicatescience.com/experiments"
},
{
"@type": "ListItem",
"position": 2,
"name": "PD-L1 regulates the development, maintenance, and function of induced regulatory T cells methods",
"item": "https://replicatescience.com/experiments/pd-l1-regulates-the-development-maintenance-and-function-of-induced-regulatory-t-cells-methods-loise-m-francisco-pmc2806460/pd-l1-regulates-the-development-maintenance-and-function-of-induced-regulatory-t-cells-mlph0y4n"
}
]
}
]