Peripheral blood mononuclear cells were isolated by density gradient centrifugation (Lymphoprep, Nycomed Pharma AS, Oslo, Norway) of buffy coats that were purchased from Sanquin blood bank (Region South-East, Netherlands). All donors gave written informed consent for the use of these buffy coats for scientific research purposes, and according to Dutch law. CD4+ T cells were enriched using the RosetteSep™ human CD4+ T cell enrichment cocktail and processed according to manufacturer’s recommendations (StemCell Technologies, Vancouver, BC, Canada). This typically resulted in a >95% purified CD4+ T cell population in the absence of CD8+ cells. To obtain high purity Treg, subsequent FACS sorting of CD4+CD25high Treg was performed using a BD FACSAria cell sorter (BD Biosciences, Erembodegem, Belgium) after labeling CD4+ cells with CD25/Pe-Cy7 (M-A251; BD Biosciences).
Cd25 pe cy7 m a251
Manufactured by BD
Sourced in Belgium, United States
The CD25/Pe-Cy7 (M-A251) is a laboratory instrument used for the detection and analysis of CD25, a cell surface antigen, using flow cytometry. It is a fluorescently labeled monoclonal antibody that binds to CD25 and can be detected using a flow cytometer equipped with the appropriate laser and filter configuration.
Automatically generated - may contain errors
Lab products found in correlation
Facsaria cell sorter, by BD (3 mentions)
Facscanto 2, by BD (2 mentions)
Rosettesep human cd4 t cell enrichment cocktail, by STEMCELL (2 mentions)
Streptavidin pe, by BD (1 mentions)
Cd4 pe cy7 sk3, by BD (1 mentions)
7 aminoactinomycin d 7 aad, by BD (1 mentions)
Human foxp3 buffer set, by BD (1 mentions)
Facsdiva, by BD (1 mentions)
Fc500 flow cytometer, by Beckman Coulter (1 mentions)
Cd4 bv510, by BD (1 mentions)
Rosettesep, by STEMCELL (1 mentions)
Transcription factor phospho buffer set, by BD (1 mentions)
Cd4 fitc rpa t4, by Thermo Fisher Scientific (1 mentions)
96 well round bottom plate, by Thermo Fisher Scientific (1 mentions)
Anti cd3, by Thermo Fisher Scientific (1 mentions)
Facsaria 3, by BD (1 mentions)
7 protocols using cd25 pe cy7 m a251
1
Isolation and Purification of CD4+ Treg Cells
2
Multiparameter Flow Cytometry Profiling
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Cells were stained with viability dye 7-aminoactinomycin D (7-AAD; BD Pharmingen) and anti-human cell surface antibodies from the following list: CD1a-BV605 (SK9; BD Biosciences),CD3-Pacific Blue (HIT3a; BioLegend), CD3-BV605 (HIT3a; BD Horizon), CD3-BV650 (UCHT1; BD Horizon), CD4-PE-Cy7 (SK3; BD Pharmingen), CD4-BV650 or -BV711 (SK3; BD Horizon), CD8-BV510 (RPA-T8; BD Horizon), CD25-PE-Cy7 (M-A251; BD Pharmingen), CD56-BV786 (NCAM16.2; BD Horizon), CD127-BV421 (HIL-7R-M21; BD Horizon), CD161-APC or -PE/Dazzle 594 (HP-3G10; BioLegend), TCR Vα7.2-FITC (3C10; BioLegend), and TCRγ/δ-1-PE-Cy7 (11F2; BD Biosciences).
For identification of NKT cells, cells were stained with PBS44 loaded human CD1d tetramers. PBS44-CD1d monomers were kindly donated by Dale Godfrey (Dept. Microbiology and Immunology, Peter Doherty Institute, University of Melbourne, Parkville, Australia). In some instances, MAIT cells were identified using 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU) loaded human MR1 tetramers. 5-OP-RU-MR1 monomers were a kind gift from the McCluskey laboratory (Dept. Microbiology and Immunology, Peter Doherty Institute, University of Melbourne, Parkville, Australia). CD1d and MR1 monomers were tetramerized by conjugation to PE streptavidin (BD Pharmingen).
For identification of NKT cells, cells were stained with PBS44 loaded human CD1d tetramers. PBS44-CD1d monomers were kindly donated by Dale Godfrey (Dept. Microbiology and Immunology, Peter Doherty Institute, University of Melbourne, Parkville, Australia). In some instances, MAIT cells were identified using 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU) loaded human MR1 tetramers. 5-OP-RU-MR1 monomers were a kind gift from the McCluskey laboratory (Dept. Microbiology and Immunology, Peter Doherty Institute, University of Melbourne, Parkville, Australia). CD1d and MR1 monomers were tetramerized by conjugation to PE streptavidin (BD Pharmingen).
3
Immunophenotypic Analysis of Regulatory T Cells
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Immunophenotypic analysis of peripheral blood was performed with an FC500 Flow Cytometer (Beckman Coulter, Brea, CA; USA) as previously described [22 (link)]. FACS analysis (FACSCanto II; BD Biosciences, Franklin Lakes, NJ, USA) of regulatory T cells in PBMCs was performed with the following: mAbs:CD4–allophycocyanin–H7 (RPA-T4) and CD25–PE–Cy7 (M-A251) (both from BD Pharmingen, Franklin Lakes, NJ, USA). Thereafter, cells were washed, fixed, and permeabilized (Human FoxP3 Buffer Set; BD Pharmingen) and were stained with FOXP3-PE (259D/C7) (BD Pharmingen). Analyses were performed with FACSDiva (BD) and FlowJo (BD) software.
4
Isolation and Injection of Human Tregs
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PBMCs where isolated from buffy coats using ficoll density gradient isolation (Lymphoprep, Nycomed-Pharma AS, Oslo, Norway). To isolate human CD4+ CD25hi Treg, first CD4+ T cells were enriched using the RosetteSepTM (StemCell™ Technologies, Vancouver, Canada) human CD4+ T cell enrichment cocktail according to the manufacturers description, followed by high purity CD4+CD25high cell sorting using a BD FACSAria cell sorter (BD Biosciences, Erembodegem, Belgium). For this purpose, cells were labeled with CD4-BV510 and CD25-Pe-Cy7(M-A251; BD Biosciences, New Jersey, USA). This typically resulted in >96% pure CD4+CD25hi cells and more than 90% of these cells were FOXP3+. Without ex vivo expansion, high purity sorted Treg (or PBS, vehicle control) were injected intradermally in the mouse skin at 4 spots closely around the human skin graft, in a total volume of 100 µl PBS.
5
Isolation of Regulatory T Cells from PBMCs
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Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation (Lymphoprep, Nycomed Pharma AS, Oslo, Norway) of buffy coats obtained from healthy blood donors (Sanquin Blood Bank, Region South-East, Netherlands) upon written informed consent, according to the Dutch law. CD4+ T cells were enriched using the RosetteSepTM human CD4+ T cell enrichment cocktail and processed according to manufacturer’s recommendations (StemCell Technologies, Vancouver, Canada). This resulted in a >95% purified CD4+ T cells and the absence of CD8+ cells. To obtain high purity Treg, FACS sorting of CD4+CD25high Treg was performed using a BD FACSAria cell sorter (BD Biosciences, Erembodegem, Belgium) after labeling CD4+ cells with CD25/Pe-Cy7(M-A251; BD Biosciences), termed as FACS-sorted Treg. More than 97% of Treg were FOXP3+ after cell sorting. Less pure MACS-isolated CD4+CD25+ Treg were prepared using human CD25 microbeads (Miltenyi Biotech, Bergisch Gladbach, Germany), according to manufacturer’s instructions. To mimic the purity of clinic grade isolation, 15–20 μL of CD25 microbeads for every ten million CD4+ cells were used. The resultant Treg were 60–80% positive for FOXP3.
6
PBMC and SFMC Stimulation and Intranuclear Staining
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PBMC and SFMC were thawed and resuspended in PBS (0,5 -1,0 x 10 6 living cells/tube).
Surface staining of CD3 (Biolegend) and CD4 (eBioscience) was performed for 25 min at 4°C.
Cells were then stimulated with 0 , 1.0 , 10 or 100 IU/ml human (h)IL-2 (Proleukin; Novartis) for 30 min at 37°C, fixated and permeabilized by using buffers from the Transcription Factor Phospho Buffer Set (BD Biosciences). Intranuclear staining of FOXP3-eF450 (PCH101), T-bet-eF660 (eBio4B10; eBioscience), CD25-PE/CY7 (M-A251) and pSTAT5-PE (pY695; BD) was performed for 50 min at 4°C. Data acquisition and analysis as above.
Surface staining of CD3 (Biolegend) and CD4 (eBioscience) was performed for 25 min at 4°C.
Cells were then stimulated with 0 , 1.0 , 10 or 100 IU/ml human (h)IL-2 (Proleukin; Novartis) for 30 min at 37°C, fixated and permeabilized by using buffers from the Transcription Factor Phospho Buffer Set (BD Biosciences). Intranuclear staining of FOXP3-eF450 (PCH101), T-bet-eF660 (eBio4B10; eBioscience), CD25-PE/CY7 (M-A251) and pSTAT5-PE (pY695; BD) was performed for 50 min at 4°C. Data acquisition and analysis as above.
7
Treg Isolation and Functional Assay
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CD3 + CD4 + CD25 + CD127 low cells (Treg) were isolated from frozen PBMC, using the FACS Aria III (BD). Antibodies used for sorting are: anti-human CD3-BV510 (OKT3), CD25-PE/Cy7 (M-A251; BD), CD127-AF647 (HCD127; Biolegend), CD4-FITC (RPA-T4; eBioscience). To check for FOXP3 expression of the sorted populations cells were fixed and permeabilized by using eBioscience Fixation and Permeabilization buffers (Invitrogen) and stained with anti-human FOXP3-eF450 (PCH101; eBioscience). Read out of proliferation is performed with the following antibodies: CD3-PerCP/Cy5.5 (UCHT1; Biolegend), CD4-FITC (RPA-T4; eBioscience), CD8-APC (SK1; BD). Total PBMC were labeled with 2µM ctViolet (Thermo Fisher) and cultured alone or with different ratios of sorted Treg (1:16, 1:8, 1:4, 1:2). Cells were cultured in RPMI1640 media containing 10% human AB serum with addition of L-Glutamine and Penicillin/Streptomycin. PBMC were stimulated by 0,1 µg/ml coated anti-CD3 (eBioscience)
and incubated for four days in a 96 well round bottom plate (Nunc) at 37˚C. After 4 days cells were stained with CD3, CD4, and CD8 for read out of proliferation by flow cytometry performed on FACS Canto II (BD Biosciences) and data was analyzed using FlowJo Software (Tree Star Inc.).
and incubated for four days in a 96 well round bottom plate (Nunc) at 37˚C. After 4 days cells were stained with CD3, CD4, and CD8 for read out of proliferation by flow cytometry performed on FACS Canto II (BD Biosciences) and data was analyzed using FlowJo Software (Tree Star Inc.).
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