Phrodo ifl green stp ester

TDP-43-MBP-His₆ was labeled with Alexa Fluor 488 C5 maleimide (Thermo Fisher) and monoclonal TDP-43 antibodies were conjugated with DyLight™ 650 NHS Ester (Thermo Fisher), both at a low (~ 0.01–0.05) labelling efficiency to not interfere with condensate formation. Following manufacturer´s instructions, TDP-43-MBP-His₆ was mixed with the Alexa Fluor reagent in a 100:1 or 20:1 protein:fluorescent dye molar ratio and kept in the dark for 2 h at RT. Monoclonal antibodies and DyLight dye were used in a 50:1 protein:fluorescent dye molar ratio and incubated for 1 h at RT protected from light. Excess dye was removed by consecutive washes with TDP-43 purification buffer (TDP-43-MBP-His₆) or PBS (monoclonal antibodies) using Amicon ultra centrifugal filters (Merck, Germany) and protein concentrations were determined. Labeled proteins were used for confocal microscopy, aggregation, and phase separation assays, respectively. For flow-cytometry based uptake assays, TDP-43-MBP-His₆ was labeled with pHrodo™ iFL Green STP Ester (Thermo Fisher) at a labeling efficiency of ~ 0.7–1.0. The labeling procedure was carried out as described above with a protein:fluorescent dye molar ratio of 1:1 and an incubation step of 1 h.

Flash red fluorescent polystyrene beads (Bangs Laboratories, Inc #FSFR004) were washed three times in sterile PBS and incubated with 25 μg/ml of recombinant Her-2 (R&D systems #10126-ER-050) for 1 h at room temperature in the dark. Beads were then washed and incubated for 1 h at room temperature in the dark with PBS with 5% fetal bovine serum (FBS) and 1:100 pHrodo iFL Green STP ester (Thermo Fischer Scientific # P36013). Beads were washed again and resuspended in PBS+5% FBS, at a stock concentration of 5 × 10⁸ beads per ml. THP-1 cells (ATCC #TIB-202) were grown in RPMI-1640 media and resuspended in 96-well plate with serially diluted antibodies and described beads at bead to cell ratio of 20:1. The cultures were incubated for 4 h at 37 ^°C and 5% CO₂. Cells were then washed twice with flow buffer (1% FBS in PBS), resuspended, and analyzed by BD Fortessa. The bead internalization is determined by the cell fraction of double positive for FITC and APC. The phagocytosis score is calculated as: GMFI (APC) ∗ % (bead internalization).