Adipose tissues including interscapular brown adipose tissue (BAT), subcutaneous adipose tissue (SAT), or epididymal visceral adipose tissue (VAT) were isolated from young or old WT mice and cut into small pieces, which digested with collagenase solution (2 mg/ml collagenase type II in RPMI 1,640 medium) at 37°C for 30 min. The samples were filtered with 40 μM strainers and centrifuged at 450 × g for 5 min at 4°C. Samples were analyzed for specific surface and intracellular markers by Beckman CytoFLEX (Beckman Coulter, United States). Antibodies used for staining were as follows: Fixable Viability Dye-APC-eFlour780 (65-0865-14, eBioscience), CD45-Violet 510 (103138, BioLegend), CD3e-Violet 605 (100351, BioLegend), IFN-γ-APC (17-7311-81, eBioscience), CD28-PE (12-0281-82, eBioscience), CD44-APC-eFlour780 (47-0441-82, eBioscience), and CD62L-eFlour450 (48-0621-80, eBioscience).
Cd28 pe
Manufactured by Thermo Fisher Scientific
CD28-PE is a fluorescently labeled antibody used in flow cytometry applications to detect the CD28 antigen on the surface of T cells. It provides a tool for the identification and analysis of CD28-positive cell populations.
Automatically generated - may contain errors
Lab products found in correlation
Cd3 pecy5, by BD (3 mentions)
Cd8 v450, by BD (3 mentions)
Rosettesep human cd8 t cell enrichment cocktail, by STEMCELL (3 mentions)
Aria 2, by BD (3 mentions)
Eflour 506, by Thermo Fisher Scientific (2 mentions)
Beckman cytoflex, by Beckman Coulter (1 mentions)
Ifn γ apc, by Thermo Fisher Scientific (1 mentions)
Ccr7 pe cy7, by BD (1 mentions)
Cd3 apc h7, by BD (1 mentions)
Cd45ra apc, by Thermo Fisher Scientific (1 mentions)
Cd4 fitc, by BioLegend (1 mentions)
Cd8 pe, by BioLegend (1 mentions)
Cd45ra fitc, by BD (1 mentions)
Pd 1 fitc, by Thermo Fisher Scientific (1 mentions)
Pharmingen pe annexin 5 apoptosis detection kit 1, by BD (1 mentions)
Cellrox green ros detection kit, by Thermo Fisher Scientific (1 mentions)
Cd57 apc, by BioLegend (1 mentions)
Ifn γ pe, by Thermo Fisher Scientific (1 mentions)
Caspglow fluorescein active caspase 3 staining kit, by Thermo Fisher Scientific (1 mentions)
Patm ser1981 pe antibody, by BioLegend (1 mentions)
5 protocols using cd28 pe
1
Characterization of Adipose Tissue Immune Cells
2
Isolation and Characterization of CD8+ T Cells
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Blood samples were collected at the Blood Centers of the Pacific in San Francisco, CA. All donors were kept anonymous and provided written informed consent. Total human CD8+ T cells were enriched via negative selection using the RosetteSep human CD8+ T cell enrichment cocktail (15063; Stemcell). The following antibodies were used to stain peripheral blood mononuclear cells or enriched CD8+ naive, CD8+CD28+ memory, and CD8+CD28− T cells: CD3-PECy5 (555334; BD Biosciences) and CD3-APC-H7 (560176; BD Biosciences), CD8-V450 (560347; BD Biosciences), CD28-PE (12–0289; eBioscience), CD45RA-APC (17–0458; eBioscience), and CCR7-PECy7 (557648; BD Biosciences). Flow cytometry was performed on an LSRII or Calibur DxP8 flow cytometer (both BD Biosciences) and was analyzed with FlowJo software. Sorting was performed on an ARIA II (BD Biosciences).
3
Isolation and Culture of CD8+ T Cell Subsets
4
Phenotypic and Functional Analysis of CD4 T Cells
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For phenotypic analysis of CD4 T cells, PBMCs were stained with CD4-FITC, CD8-PE, CD57-APC (BioLegend, San Diego, CA), CD28-PE (Invitrogen, Carlsbad, CA), CD45RA-PerCP710, PD1-FITC (eBioscience, San Diego, CA), or isotype control antibodies. To quantify cell apoptosis, PBMCs were stained with CD4-A647 and CD45RA-FITC for naïve or memory cell populations, and then stained with Annexin V-PE and 7-AAD using BD Pharmingen™ PE Annexin V Apoptosis Detection Kit I (BD Biosciences, San Jose, CA). CD4+ T cells were also stained for caspase-3 expression following a protocol from CaspGLOW™ Fluorescein Active Caspase-3 Staining Kit (Invitrogen). Levels of reactive oxygen species (ROS) in CD4 T cells were measured using the DCFDA-based Cellular ROS Detection Kit (Abcam, Cambridge, MA) or CellROX Green ROS Detection kit (ThermoFisher Scientific, Waltham, MA) according to manufacturer's protocol. For intracellular staining, the cells were fixed and permeabilized with Foxp3 Transcription Factor Staining Buffer Set (eBioscience), and stained with pATM (Ser1981)-PE antibody (BioLegend), pCHK2 (Thr68)-PE antibody, γH2AX-PE (eBioscience), IL-2-PE, and IFN-γ-PE (Invitrogen). Flow cytometry was carried out as described previously (19 (link), 20 (link)).
5
Isolation and Culture of CD8+ T Cell Subsets
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Blood samples were collected at the Vitalant in San Francisco, CA. All donors were age 50 or older, provided written informed consent, and were de-identified. Total human CD8+ T cells were enriched using the RosetteSep Human CD8+ T Cell Enrichment Cocktail (Stemcell Technologies, 15063). Then, the following antibodies were used to stain CD8+CD28+ and CD8+CD28- T cells: fixable viability dye eFlour506 (Invitrogen, 65–0866-14), CD3-PECy5 (BD Biosciences, 555334), CD8-V450 (BD Biosciences, 560347), and CD28-PE (Invitrogen, 12–0289-42). Sorting was performed on an ARIA II (BD Biosciences). The gating strategy is illustrated in Supplementary Fig. 1b .
T cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 2 mM l-glutamine, and penicillin–streptomycin. Lys05 was used at 5 μM for 14 h.
T cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 2 mM l-glutamine, and penicillin–streptomycin. Lys05 was used at 5 μM for 14 h.
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