Legendscreen human pe kit

LEGENDScreen™ Human PE kit (Biolegend, Cat#700007) was used to screen the cell surface proteome. Fresh SVC from subcutaneous liposuction aspirates and PBMC from buffy coat blood were used in the screening as previously described (33 (link)). In brief, the tissue-derived and blood-derived cells were CD45 barcoded and stained with a backbone antibody panel allowing identification of the cells of interest. The cells were added to the plates provided in the LEGENDScreen™ kit and the staining was performed according to the manufacture protocol. Plates were run on an 18-color flow cytometer (LSR Fortessa, BD Biosciences) with 407, 488, 561 and 640 lasers using the BD FACSDiva™ Software (BD Biosciences). Flow cytometry data was analyzed using FlowJo v10 (Treestar, USA). Two manual gating approaches were performed to identify the NK cell subsets, a “broad” gating followed by a “fine” gating. Several adipose-enriched proteins identified from both gating approaches were validated for expression in additional adipose tissue samples, and a selection of these proteins was included in the 27-parameter flow cytometry experiment.

On day 47, HP organoids were dissociated with Accumax (Innovative cell technologies, #AM105) to single cells and stained using a LEGENDScreen Human PE Kit (BioLegend, #700001), which contains PE-conjugated 332 surface marker antibodies and 10 isotype controls. This and the following procedures were conducted in low-cell-adhesion V-bottom 96-well plates (1×10⁵ cells/1 surface marker antibody or isotype control/well). After washing via centrifugation, cells were fixed and permeabilized using an IntraStain kit (Dako, #K2311) and incubated with an FITC-conjugated mouse anti-cytokeratin monoclonal antibody (1:30; clone CK3-6H5; Miltenyi Biotec, #130-080-101, RRID: AB_244191), 5 µg/ml Hoechst 33342, and HCS CellMask Deep Red Stain (Invitrogen, #H32721) for 20 min at room temperature (RT). Finally, cells were washed in Cell Staining Buffer (BioLegend, #420201) and transferred to 96-well imaging plates (CellCarrier-96; PerkinElmer, #6005550). Fluorescence images from each well were acquired and analyzed using the Opera Phenix high-content imaging system (PerkinElmer, RRID: SCR_021100) to determine the percentage of cell populations automatically (1700–11200 Hoechst⁺ nuclei were detected per well). CellMask signals were used for segmentation of cell areas. Cells stained with isotype controls were used as negative controls to determine positive staining for surface markers.