Bx43 fluorescent microscope

Cells were fixed with 4% paraformaldehyde, and rinsed twice with 1× PBS. Primary antibodies included rabbit CD86 (1 μl/ml; Abcam, Cambridge, MA), rat CD206 (5 μl/ml; Abcam), rabbit Beta III Tublin (5 μl/ml, Abcam), rabbit anti-cleaved caspase-3 (0.5 μl/ml; Abcam), and mouse NeuN (10 μl/ml; Millipore, Billerica, MA). In addition, cell nuclei were stained with DAPI (0.5 μl/ml; Life Technologies, Carlsbad, CA). Fluorescently tagged secondary antibodies (Invitrogen) were visualized with an Olympus BX43 fluorescent microscope with a CellSens Standard imaging program. Five randomly selected images were taken per sample and each image was qualitatively assessed for overall fluorescence. NeuN-positive cells were manually counted using NIH ImageJ (Bethesda, MD).

Single nuclear capture and sequencing were performed on the ICELL8 single-cell platform (Wafergen Biosytems). ICELL8 platform comprised of a multi-sample nano-dispenser that precisely dispensed 50 nl of the single-nuclei suspension into an ICELL8 nanowell microchip-containing 5184 wells (150 nl capacity). Assuming a Poisson distribution frequency for the number of cells per well, about 30% of the nanowells were expected to contain a single nucleus under optimal conditions. Automated scanning fluorescent microscopy of the microchip was performed using an Olympus BX43 fluorescent microscope with a robotic stage to visualize wells containing single nuclei (see Table 1 for single-cell capture number across different experimental repeats). The automated well selection was performed using the CellSelect software (Wafergen Biosystems), which identified nanowells containing single nuclei and excluded wells with >1 nuclear, debris, nuclei clumps or empty wells. The candidate wells were manually evaluated for debris or clumps as an additional QC.

Membrane potential was measured using a potentiometric fluorescent probe, MitotrackerCMXRos (Invitrogen, UK) that sequesters in mitochondria dependant on proton gradients. Animals were grown to young adult stage with or without respective SAA seeded on plates as described above. A 940 μM stock of the probe was made up in 100% DMSO and diluted to 5 μM (0.5% DMSO) with 40 μL placed into black tubes. Approximately 40 animals were picked into each 40 μL tube and incubated at 20 °C for 1 h in the dark. Before imaging, animals were washed 3× with M9 to remove any residual dye on the outer cuticle of the animal. Animals were then pipetted onto slides and immobilised with a coverslip and imaged in dim lighting. Images were taken using a ×40 objective on an Olympus BX43 fluorescent microscope with green light excitation, with exposure set to 500 ms for all conditions. For quantification, the integrated density was measured and normalised to the area of measurement.

The TUNEL assay was performed according to the manufacturer’s instructions (TUNEL Apoptosis Detection Kit [Alexa Fluor 488], Yeasen, #40307ES20) to detect cell death in the synovial membrane. The assay used the green channel at 488 nm. DAPI was applied as a nuclear counterstain in the blue channel at 461 nm. Images were taken with an Olympus BX43 fluorescent microscope and Olympus DP73 digital camera at ×400 magnification with cellSens software (Olympus). Exposure settings were adjusted to minimize oversaturation.

Samples were fixed with 10% buffered formalin (cat. no. SF1004, Thermo Fisher Scientific, Waltham, MA, United States) and stained with 5 μM SYTOX orange (cat. no. S34861, Thermo Fisher Scientific, Waltham, MA, United States) and NucBlue^TM Fixed Cell ReadyProbes^TM Reagent (DAPI, cat. no. R37606, Thermo Fisher Scientific, Waltham, MA, United States) as described (Minden-Birkenmaier et al., 2020 (link)). Briefly, samples were sequentially incubated with each stain for 5 min at room temperature. Three washes with 1 × phosphate-buffered saline for 5 min each were performed between each step. Cells and NETs were visualized on an Olympus BX43 fluorescent microscope.

The preliminary characterization of LC and LC-QD flows in microfluidic confinement was performed by digital optical microscopy using a Levenhuk D320 optical microscope (Levenhuk, Tampa, FL, USA). Microscopy images were captured at 100× magnification using a ToupCam E3ISPM08300KPB camera (Touptek, Hangzhou, China).
The orientation behavior of the LC material and LC-QD composites in microfluidic flows were studied by polarized optical microscopy (POM) using an Olympus BX51 microscope (Olympus, Tokyo, Japan) equipped with a high-precision Linkam heating system. Microscopy images were captured at 100× and 500× magnification using a ToupCam E3ISPM08300KPC camera (Touptek, Hangzhou, China).
The luminescent properties of LC-QD composites in microchannels were studied by an Olympus BX43 fluorescent microscope (Olympus, Tokyo, Japan). Microscopy images were captured at 100× magnification using a ToupCam E3ISPM05000KPA camera (Touptek, Hangzhou, China).
Polarized and fluorescent microscopy images were processed by Matlab 2021b software. For processing, microscopy images were taken at identical microscope settings. The luminance components of polarized microscopy images and the red color components of fluorescent microscopy images were extracted and processed by the pre-developed Matlab script.

Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay was conducted to assess the level of apoptosis in GTN cells. Briefly, GTN cells were seeded onto coverslips in 24-well plates (2×10⁴ cells/well) and incubated overnight before addition of MTX. After MTX treatment (10 μM for MTX-resistant sublines; 3 μM for parental cell lines) for 96 hours, GTN cells grown on coverslips were fixed with 4% paraformaldehyde and then permeabilized with 0.1% Triton-X 100 in PBS. After being washed by PBS, cells were incubated with reaction mixture for 60 min at 37°C. Coverslips were then mounted and analyzed under an Olympus BX43 fluorescent microscope. The percentage of cells identified by TUNEL in each section was determined in four randomly selected fields (10× objective). Data are presented as mean ± SD from four independent experiments.