Cellstar multiwell culture plates

BioTek powerWaveTM XS (BioTek Instruments, (Winooski, VT, USA), FP-6200 (JASCO, Easton, MD, USA) and Infinite^® 200 PRO multimode (TECAN, Deutschland GmbH, Crailsheim, Germany) microplates readers, convection oven (Romag S.A, Barcelona, Spain), UN 500 universal oven (Memmert, Schwabach, Germany) and Telstar Lyobeta-15 lyophilizer (Telstar, Madrid, Spain) were used for analyses. Forcell culture assays, a Neubauer cell counting chamber (0.100 mm depth, 0.0025 mm²) (Paul Marienfeld GmbH & Co., KG, Lauda-Königshofe, Germany), an incubator (BINDER GmbH, Tuttlingen, Germany), a Thermo Scientific™ MSC-Advantage™ class II biological safety cabinet (Thermo Fisher Scientific, Waltham, MA, USA), an autoclave (3870EA, Tuttnauer, Hauppauge, NY, USA), a TR400-SW TRINO microscope (VWR, Vienna, Austria), CELLSTAR^® multiwell culture plates and standard cell culture flasks (Greiner Bio-One GmbH, Kremsmuenster, Austria) were used.

Primary human macrophages were differentiated from peripheral blood monocytes cells (PBMC). After isolation of PBMC’s in a density gradient medium, cells were split into 12-well culture plates (Greiner CELLSTAR multiwell culture plates, 0.4 × 10⁶ cells/well) and incubated at 37 °C, 5% v/v CO2 and 95% v/v O₂ for 1 h in a free serum medium (RPMI 1640, 2 mM l-glutamine, 1% Pen/Strep, Biological Industries). Non-adherent contaminated cells were removed and remaining monocytes differentiated to human microglia like cells for 10 days with recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF, 10 ng/ml; R&D systems) in a full RPMI medium (RPMI 1640, 10% FBS, 2 mM l-glutamine, 1% Pen/Strep) and incubated at 37 °C, 5% v/v CO₂ and 95% v/v O₂.

BV-2 (mouse microglia, ICLC ATL03001) cells were split into 12-well culture plates (Greiner CELLSTAR multiwell culture plates) on the day before the experiment. A total of 0.3 μM aggregated TTR conjugated with Alexa 488, pre-incubated with 0.1 0.2, 0.5, 1, 2, 3 and 4 μg/mL of Ab-A or 0.1, 1, 3 and 4 μg/mL human control IgG for 2 h at room temperature, were added to cells with culture media (RPMI 1640, 10% FBS, 2 mM L-Glutamine, 1% Pen/Strep). The conditioned medium was also pre-incubated with Ab-A (without aggregated TTR) for 2 h as a control. The cells were then incubated at 37 °C for 24 h and harvested. Extracellular and cell surface-aggregated TTR conjugated with Alexa 488 in cell pellets was quenched by the incubation of 0.4% trypan blue in PBS (pH 4.4) for 1 min. Flow cytometry (FL1—blue laser (488 nm)) was used to measure aggregated TTR (conjugated to ALEXA fluor 488) uptake, indicated as the relative geomean fluorescence intensity (MFI).