Eclipse te2000 microscope

THP-1 cells, a human monocytic cell line, were obtained from the American Type Culture Collection (ATCC, #TIB-202™, Manassas, VA, USA) and maintained in RPMI-1640 medium (Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS) until experimentation. Cells that had undergone no more than 20 passages were incubated in medium containing phorbol 12-myristate 13-acetate (PMA; final concentration, 150 nM) in 24-well plates for 24 h at 37 °C in a humidified incubator (5% CO₂, 95% air) to induce differentiation into macrophages. Differentiated and adherent macrophages were then rinsed with warm phosphate buffered saline (PBS) and incubated with 400 µL of fresh RPMI-1640 medium containing 1% FBS, 50 µL of acetylated low-density lipoprotein (acLDL) (1 mg of protein/mL in PBS), and 50 µL of PBS or ferrous ion (final 60, 120 µM of ferrous ion in ddH₂O) for 48 h at 37 °C in a humidified incubator. After incubation, cells were washed with PBS three times and then fixed in 4% para-formaldehyde for 10 min. Next, fixed cells were stained with oil-red O staining solution (0.67%) and then washed with distilled water. THP-1 macrophage-derived foam cells were then observed and photographed using a Nikon Eclipse TE2000 microscope (Tokyo, Japan) at 600× magnification.

Microscopic analysis of CD19, Igκ, and Igλ expression in human B cells was performed by fluorescent microscopy and ImageStream. Human PBMCs were stained with anti-CD19-APC, anti-Igλ-PE, and anti-Igκ-FITC (for cytospin) or anti-Igκ-BV421 (for ImageStream). Stained cells were washed in staining buffer PBS, 1% BSA, 0.1% NaN₃ and further processed for analysis. For microscopy, 1x10⁶ stained cells, resuspended in 150 μl of staining buffer, were loaded in a cytospin centrifuge and spun at 1,500 rpm for 5 minutes into each microscope slide. After drying the slides in air for 10 minutes, one drop of cytoseal mounting media was added and a cover slip applied. Cytospin slides were analyzed, and images collected using an Eclipse TE 2000 microscope and NIS Elements version 4.2 software (Nikon). Stained cells were also analyzed by ImageStream (Amnis, Seattle, WA) at a concentration of 1x10⁶ cells/ml and under blinded conditions. ImageStream data were analyzed using IDEAS 6.2 Software (Amnis).

For droplet formation without crowding agents (Figure 2A, up panel, Figure 2B, C , and D), proteins were diluted to various concentrations in the buffer containing a final concentration of 25 mM Tris-HCl, pH 7.4, 75 mM KCl at room temperature. For droplet formation in the presence of crowding agents (all the other in vitro phase separation experiments), proteins were diluted from a stock solution into buffer containing a final concentration of 5 % dextran, 25 mM Tris-HCl, pH 7.4, 150 mM KCl at room temperature. Proteins were added as the last component to induce uniform phase separation. To observe the propensity of RNA to partition into the condensates, we resuspended RNA in RNase-free water at indicated concentrations. The droplet formation of purified mGFP-YBX1 and Cy5 labeled miRNAs (100 nM miRNAs together with 10 ng/µl total RNA, which is about 100 nM) was induced in LLPS buffer (5 % dextran, 25 mM Tris-HCl, pH 7.4, 150 mM KCl, 1 mM MgCl₂ (to stabilizes the RNA secondary structure)). The samples were mixed in a microtube and applied to a coverslip-bottom in 35 mm dishes (MatTek P35G-1.5–14 C). After all the droplets had settled to the bottom, images were taken using an ECLIPSE TE2000 microscope (Nikon) with a 100 x oil-immersion objective.

Live-cell images were taken twenty-four hours after transfection using Yokogawa VSU-10 spinning disc confocal system attached to Eclipse TE-2000 microscope (Nikon, Tokyo, Japan). Images for mitochondria and lysosomes, as well as Drosophila brains, were taken using Zeiss LSM 780 laser scanning confocal microscope (Zeiss, Oberkochen, Germany). Images for Congo red staining were taken using Zeiss LSM 700 laser scanning microscope (Zeiss). All images were analyzed using Zen software (Zeiss) and Fiji software [48 (link)].