Orca flash4.0 v2

Chlormethylbenzamido-1,1-dioctadecyl-3,3,3',3'-tetramethylin-docarbocyamine (CM-Dil) is a type of tracer that is commonly used for labeling cellular membrane. Cells were centrifuged at 1,000 rpm for 5 min at 4°C and washed with PBS twice, and then incubated with 5 µl CM-Dil dye (ThermoFisher, Cat# C7001, USA) at 37°C for 5 min and 4°C for 15 min. Under the excitation wavelength, the cell membranes displayed orange-red color when excited by green light.
To detect the cytotoxicity of NK cells after AZA treatment at the single-cell level, the microfluidics chips were designed for single-cell capture as previously described (Liang et al., 2018 (link); Yan et al., 2018 (link)). AML cells with CM-Dil staining were injected into the chip and fixed in one inlet. After AZA treatment, NK cells were injected from another inlet and the AML cells and NK cells were fixed in the middle of the chips and contacted with each other. The dynamic changes in cell morphology at different time points were observed by microscopy (NIKON, ECLIPSE TiU) and the images were acquired with a sCMOS camera (Hamamatsu, ORCA-Flash 4.0 v2).

The lattice light sheet microscope located at the Advanced Imaging Center (AIC) at the Janelia Research Campus of the Howard Hughes Medical Institute (HHMI) (Chen et al., 2014 (link)) was used. HUVECs stably expressing dTomato-2xrGBD and mTurquoise2-CaaX were cultured on fibronectin-coated 5 mm round glass coverslips (Warner Instruments, Catalog # CS-5R) for 2 days. Cells were imaged at 37°C in the presence of 5% CO₂ in HEPES buffer (132 mM NaCl₂, 20 mM HEPES, 6 mM KCl₂, 1 mM MgSO₄•7H₂O and 1.2 mM K₂HPO₄•3H₂O at pH 7.4), supplemented with 1 mM CaCl₂, 0.5% Albuman (Sanquin Reagents, The Netherlands) and 1 g/l D-glucose. Illumination was undertaken using 445 nm and 560 nm diode lasers (MPB Communications), acousto-optic tunable filter (AOTF) transmittance and 100 mW initial box power and an excitation objective (Special Optics, 0.65 NA, 3.74-mm WD). Fluorescence detection was done via a detection objective (Nikon, CFI Apo LWD 25XW, 1.1 NA) and a sCMOS camera (Hamamatsu Orca Flash 4.0 v2). Point-spread functions were measured using 200 nm tetraspeck beads (Invitrogen cat# T7280) for each wavelength. Data was deskewed and deconvolved as described previously (Chen et al., 2014 (link)).

For larger amplitude force-displacement experiments we used an in-house customized micromanipulation setup to allow uniaxial indentation of C. elegans with a microforce sensing probe (Elmi et al., 2017 (link)). Treated animals were mounted on a 2% agarose pad on top of a microscope slide (Figure 3B). To prevent motion during indentation, animals were glued (Dermabond glue, Suturonline.com) on the side to the edge of a coverslip fixed on top of the agarose pad before immersion in M9 buffer. The sample imaged using an upright widefield fluorescence microscope (BX51WI, Olympus) with 20x/1.0 water immersion objective lens (LUMPlanFL N, Olympus) fluorescence filter cube (Semrock) and a sCMOS camera (Orca-Flash4.0 v2, Hamamatsu Photonics). The body of each animal was indented using a microforce sensing probe (FT-S100, FemtoTools) fitted with a tungsten tip. The position of the probe was controlled using a motorized 4-axis stage system (ECS series, Attocube), which allowed precise positioning of the tip within (x, z) and perpendicular (y) to the focal plane of the microscope, as well as adjustment of the in-plane tilt. Animals were mounted on a separate kinematic stage system decoupled from the microscope body and the probe.

The cells on glass coverslips were fixed with phosphate-buffered saline containing 4% paraformaldehyde for 30 min and then permeabilized with 0.1% Triton X-100 for 15 min at room temperature. The fixed cells were stained with primary antibodies at room temperature for 2 h and then incubated with Alexa Fluor 488– or Alexa Fluor 546–conjugated secondary antibodies for 2 h. The primary antibodies used in this study are as follows: anti-vimentin (V9) (1:200), anti-Rac1-GTP (1:200), anti-E-cadherin (1:250), and anti-Tiam1 (1:50). Coverslips were mounted in DAPI Fluoromount-G (Southern Biotech). The images were acquired using a Zeiss Axio Imager M2 microscopy system equipped with a Plan Apochromat 10×/NA 1.4 or 20×/NA 1.4 immersion objective and a camera (ORCA-Flash 4.0 V2; Hamamatsu).