Flow cytometer s100exi

The following antibodies were used for detection by flow cytometry as previously described [22 (link)]: LIFR, gp130, PD-L1, and detection of activation markers was performed similarly using the conjugated antibodies CD14-APC, CD16-APC, CD150-APC, and CD163-APC (Table S1).
For the detection of intracellular JunB and Iba-1, microglia cells were fixed with methanol for 20 min at −20 °C, then washed twice with RPMI 1640 supplemented with 10% FCS, and processed as previously described [17 (link)].
Antigen expression was determined using an S100EXi flow cytometer (Stratedigm, Inc., San Jose, CA, USA) with CellCapTure software v4.1 (https://stratedigm.com/cellcapture/ accessed on 5 February 2023) (Stratedigm, Inc.) and FlowJo v10 (FlowJo, Ashland, OR, USA). Dead cells were gated out from the analysis.

Antibody-dependent neutrophil-mediated phagocytosis was assessed by the measurement of the uptake of antibody-opsonized, antigen-coated fluorescent beads by primary neutrophils26 (link). Biotinylated A244 gp120 was used to saturate the binding sites on 1 μm fluorescent neutravidin beads (ThermoFisher). Excess antigen was removed by washing the beads, which were then incubated with animal Ab samples for 20 min at 37 °C. Leukocytes were isolated from blood collected from HIV-seronegative donors by ACK lysis of red blood cells. Following opsonization, the freshly isolated leukocytes were added, and the cells were incubated for 1 h at 37 °C to allow phagocytosis. The cells were then stained for CD66b (BioLegend #305112; 1 μl per test) to identify neutrophils and fixed, and the extent of neutrophil phagocytosis was measured via flow cytometry (gating on CD66b-positive cell) on a Stratedigm S100EXi flow cytometer equipped with high-throughput sampler. The data are reported as a phagocytic score, which takes into account the proportion of effector cells that phagocytosed and the degree of phagocytosis (integrated MFI: frequency × MFI).

Cells were seeded
in a 24-well plate (100,000 cells per well) with 0.5 mL culture media
and incubated for 24 h. A solution of Alexa-488 labeled compounds
(75 μL, 1.5 mg mL^–1) in culture media was
dosed to the cells in triplicate under three experimental conditions:
incubation at 37 °C for 3 or 24 h and incubation at 4 °C
for 3 h. For the 4 °C experiment, the 24-well plate and sample
solution were cooled on ice for 10 min prior to the dosage of the
compound and kept on ice for the duration of incubation. After incubation,
the culture media was then removed, and the cells were washed twice
with cold PBS, treated with Trypsin, and incubated for 10 min to harvest
them. A solution of bovine serum albumin (BSA, 10%, 0.3 mL) was added
to each well, transferred to a 96-well plate, and centrifuged at 350
G for 5 min, after which the supernatant was discarded, and the cells
were resuspended in 10% BSA solution. Samples were analyzed using
a S100EXi flow cytometer (Stratedigm), equipped with 405, 488, 552,
and 640 nm solid-state lasers. Forward and side scatter gates were
used to exclude debris and dead cells using a viability dye (propidium
iodide). The mean fluorescence intensity for a population of 10,000
cells was determined with Flowjo v8 for each experimental condition
in triplicate.