Inspire software

Phagocytosis assay was performed as described previously [53 ]. Briefly, isolated monocytes were stained with PKH67 (Sigma-Aldrich) according to the manufacturer's instructions and differentiated to macrophages by 20 ng/ml Macrophage-Colony Stimulator Factor (M-CSF) (R&D Systems) in X-VIVO 10 medium (Lonza) supplemented with 10% autologous serum. MOLM-13 cells were stained with PKH26 (Sigma-Aldrich) following the manufacturer's instructions and incubated in a 1:2 E:T ratio with licMABs or mAb concentrations ranging from 0.01 nM to 100 nM for 2 h. Polybead^® Carboxylate Red-Dyed Microspheres of 6 μm (Polysciences) were used as a positive control and incubation either at 4°C or at 37°C in the presence of 10 μM Cytochlasin D (Sigma-Aldrich) served as a negative control. Cells were harvested, measured by imaging flow cytometry using an ImageStream^®X Mark II instrument (Merck Millipore, Billerica, Massachusetts, USA) and analyzed with IDEAS^® and INSPIRE^® Software (Merck Millipore, Billerica, Massachusetts, USA). The maximum phagocytosis value was set to 100% and all data points were normalized accordingly. Mean values and standard errors of triplicates were calculated and plotted.

BSLs purified from perfused brain tissue of moribund C57BL/6 mice on day 6 post-infection were stained with TCRβ-FITC, CD3ε-PE, CD4-PerCP, CD4-PerCP/Cy5.5, CD8-APC/Cy7, CD11b-APC, CD14-PE/Cy7, and F4/80-Brilliant Violet 421™ antibodies purchased from Biolegend (San Diego, CA) and fixable viability dye eFluor^®506 (eBioscience, San Diego, CA). An Amnis^® ImageStream^®X MKII (Amnis Merck-Millipore, Seattle, WA) equipped with five lasers (355, 405, 488, 561, and 642nm) was used for acquisition. A minimum of 30,000 events were collected at 40X magnification using the INSPIRE^® software (Amnis Merck-Millipore, Seattle, WA). Analysis was performed using IDEAS® 6.1 software (Amnis Merck-Millipore, Seattle, WA). Focused events were first gated using the Gradient RMS feature. Singlets were then gated by aggregate discrimination using aspect ratio and area. Analysis was then performed on gated live CD11b^highCD14⁺F480⁺TCRβ⁺CD3ε−CD4−CD8− cells.

Chondrocytes were detached from culture plates by use of non-enzymatic cell dissociation buffer (5 mM EDTA in PBS). Cells were then suspended in FACS buffer (5 mM EDTA, 3% FBS in PBS) and labeled with AnnexinV-PE (Apoptosis Detection Kit, eBioscience^TM, Waltham, MA, USA) and 7-amino-actinomycin staining (7AAD) (eBioscience^TM, Waltham, MA, USA). ANX V/7AAD double staining is a convenient way to discriminate early apoptosis from late apoptosis and necrosis. In particular, during early chondrocyte apoptosis, phosphatidylserine is translocated to the outer layer of the cell membrane, and it binds to the transmembrane protein ANX V, whose on-surface staining can be therefore used to detect apoptosis [43 (link)].
Analysis was performed in LSRII cytometer using FACS Diva6 (Becton Dickinson, Franklin Lakes, NJ, USA) and FlowJoX software (Ashland, OR, USA), version 9, for data processing. Imaging flow cytometry was performed using the ImageStream^®X Mark II Imaging Flow Cytometer (Merck Millipore, Billerica, MA, USA) with INSPIRE software (Merck Millipore, Billerica, MA, USA), then evaluated in IDEAS software (Merck Millipore, Billerica, MA, USA) version 6.0.

Stained cells (2 × 10⁷ cells/mL) in 50 μL of PBS (−) were acquired using imaging flow cytometry (ImageStreamX Mark II, Merck, Darmstadt, Germany) with two cameras, a 60 × magnification objective lens (1 pixel = 0.3 μm × 0.3 μm), and 12 detection channels controlled by INSPIRE software (Merck). The raw image files were analyzed using IDEAS software (Merck). Samples with a bright field (BF) area lower than 60 pixels (5.4 μm²) were gated out to eliminate debris. A minimum of 5,000 events, primarily involving the minor cell population (CD33⁺⁺ or CD19⁺), were collected per sample. The raw image files (.rif) were converted to data analysis files (.daf) using the compensation matrix file (.ctm). For the SMN spot analysis, we chose the nomenclature of “SMN spot” (see Supporting S1 and S2 Figs online). The definition can be summarized as follows: 1) The SMN spot was detected within the nuclear area. 2) The intensity of the maximum bright area of the SMN spot was more than 5.5 times that of background. 3) The areas of SMN spots were defined as ranging between 0.34 and 8.55 μm². 4) The shapes of SMN spots were defined based on the minor axis to the major axis ratio being 0.4 to 1.0. The details of the analysis protocol, the analytical procedure, and the algorithm design used for detecting SMN spots are described online in Supporting S1 and S2 Figs.

ROS production was determined based on the CellROX oxidation procedure (Molecular Probes). Briefly, 1 × 10⁶ intra-abdominal leukocytes were incubated with 5 μM of CellROX reagent, followed by 30 min incubation at 41°C. Leukocytes were washed twice with PBS^−/− and fixed in 1% formaldehyde. Data were acquired on an ImageStream MKII multispectral imaging flow cytometer (Amnis Corporation, EMD Millipore, Seattle, WA, USA) and analyzed using INSPIRE software (Amnis Corporation, EMD Millipore; Seattle, WA, USA).