Accutase cell detachment solution

Whole blood from apheresis was obtained from healthy donors at Boston Children’s Hospital’s (BCH) Plasma Donation Center. Because donor samples are completely de-identified at BCH prior to handling by research personnel and use in the laboratory, there is no IRB associated with the protocol. PBMCs were isolated via Ficoll gradient (Lymphoprep, StemCell 07801). CD14+ monocytes were then isolated from PBMCs using the MojoSort Human CD14 Selection Kit (Biolegend 480025) according to manufacturer protocol and plated at 5 × 106 cells per well in a 6-well plate in RPMI supplemented with 10% (v/v) FBS, penicillin/streptomycin, and 50 ng/ml M-CSF. On days 2, 4, and 6 after plating, culture media was refreshed. On day 6, cells were lifted from the plate using cold PBS and Accutase cell detachment solution (StemCell 07920) and replated at 2.5 × 104 cells/well in a 96 well plate.
On day 7, cells were given fresh media and treated with either nothing, 100 ng/ml LPS (Sigma–Aldrich, L2654), 20 ng/ml IL-4 (Peprotech, 200-04), or 100 μg/ml PAS polymers or β-glucans. After 24 h of incubation, cells were lifted from the plate with cold PBS and Accutase cell detachment solution and stained for CD68 (StemCell 60105FI) and either CD80 (Biolegend 333611), CD163 (Biolegend 305221), or CD206 (R&D Systems FAB25342P). Cells were then analyzed via flow cytometry (Attune, ThermoFisher Scientific).

Cellular superoxide anions were detected and quantified using dihydroethidium (DHE, D1168; Thermo Fisher Scientific) as a previously reported procedure (Wang et al., 2019 (link)). Briefly, neurospheres were dissociated into single cells with Accutase™ cell detachment solution (No. 07920; StemCell Technologies). After washing three times with PBS, the dissociated single cells were pellet and resuspended with prewarmed PBS containing 10 μM DHE. After incubating for 15 min in the dark, cells were precipitated and washed three times with PBS. Lastly, an equal number of cells (1 × 10⁵ cells/well) was added to the 96-well plate. The fluorescence intensity was measured using a Varioskan LUX multimode microplate reader (Thermo Fisher Scientific) with excitation and emission at 510 and 600 nm, respectively.

Macrophages were collected by Accutase cell detachment solution (STEMCEll Technology, Vancouver, Canada). M1 and M2 phenotypes were detected by flow cytometry. In this process, cells were stained with fluorescence-conjugated monoclonal antibodies including CD14-PE-Cy7, CD68-PE, CD80-BV510, HLA-DR-Percp/Cy5.5, CD163-BV421, and CD206-APC, together with LIVE/DEAD Fixable Dead Cell APC/Cy7 (all from Thermo Fisher Scientific) according to the manufacturer’s protocol. For the expression of PFKFB3 in different macrophage subtypes, the primary rabbit anti-human PFKFB3 and goat anti-rabbit secondary antibody conjugated with Alexa Fluor 488 (Jackson Laboratory, Bar Harbor, ME, USA) were employed to perform the intracellular staining with the fixation/permeabilization solution (BD Bioscience) according to the protocol of manufacture. The fluorescence was detected by the BD LSRFortessa flow cytometer (BD Bioscience), and the data were analyzed by FlowJo X (BD Bioscience).