Nucblue live readyprobe

Cells were fixed by adding 4% paraformaldehyde (PFA) solution into the channels and incubated for 15 min at RT. For vSMCs on PDMS, a 100 µL droplet of 4% PFA was carefully added on top of the cells. Cells were then permeabilized for 10 min at RT using 0.1% Triton X-100 in PBS (-). Subsequently, cells were blocked for 1 h at RT using 1% bovine serum albumin (BSA) in PBS (-). Primary antibodies were diluted 1:200 in 1% BSA, injected into the channels, and incubated overnight at 4 °C. The primary antibodies used were against CD31 (PECAM1, mouse, M0823, Agilent Dako, Santa Clara, CA, USA) and MAP2 (mouse, ab11267, Abcam, Cambridge, UK) to stain ECs and neurons, respectively. For staining of F-actin in vSMCs, phalloidin was used (ActinGreen 488 ReadyProbes, R37110, Thermo Fisher Scientific). Samples were washed 3 times with PBS (-) afterward. Secondary antibody (A-21203, Thermo Fisher Scientific) was then diluted 1:300 in 1% BSA, injected into the channels and incubated at RT for 2 h. For the neurons in the chip with channels of different heights, an overnight incubation at 4 °C was performed. Cell nuclei were stained using Hoechst 33342 (NucBlue Live ReadyProbes, R37605, Thermo Fisher Scientific). Samples were stored in PBS (-) in the dark at 4 °C until imaging.

PLVs with or without the adjacent SSV were processed for whole mount immunofluorescent microscopy, as previously described [22 ]. Briefly, after dissection the vessels were fixed in 10% normalized buffered formalin (NBF) for 30 min, and then washed in 0.1% Triton X-100 (Millipore Sigma Cat# X100) / 1× TBS (Bio-Rad Cat# 1706435) solution (3×, 10min) rocking at room temperature. The vessels were then permeabilized overnight in 0.3% Triton X-100 / 1× TBS solution rocking at 4 °C. The vessels were blocked in 5% normal goat serum (Thermo Fisher Scientific Cat#50062Z) / 0.3% TritonX-100 / 1× TBS solution rocking at room temperature for 1 h. The vessels were incubated overnight rocking at 4 °C in primary antibody solution diluted in 5% normal goat serum / 0.3% TritonX-100 / 1× TBS; anti-alpha smooth muscle actin (αSMA) Alexa Fluor 488 conjugated antibody (Thermo Fisher Scientific Cat# 53-9760-82) was used at 1:100 dilution. To wash the primary antibody, the vessels were incubated with 0.1% TritonX-100 / 1× TBS (3×, 10 min) then mounted on a microscope slide with 1 drop of ProLong Gold Antifade Mountant (Thermo Fisher Scientific Cat# P36930) and NucBlue Live ReadyProbes (Hoechst 33342 formulation; Thermo Fisher Scientific Cat# R37605). The slides were then imaged by an Olympus VS120 slide scanner or Nikon A1R HD confocal microscope.

Immunofluorescence study, and image acquisition were performed with some modifications from previous studies [27 (link)]. Briefly, cells were cultured on glass slide chamber with phenol red free RPMI at appropriate density. On the day of immunofluorescence experiment, cells were washed with PBS, and medium was changed to fresh phenol red free RPMI with or without 3-NA. After 5 h incubation at 37 °C/5% CO₂, 5, 5′, 6, 6′-tetrachloro-1, 1′, tetraethylbenzimidazolocarbocyanine iodide (JC-1, mitochondrial membrane potential detection indicator, ThermoFisher Scientific) and NucBlue® Live ReadyProbes (Nuclear staining solution, ThermoFisher Scientific) were added to each well, and incubated for 30 min. Cells were washed with PBS and fresh phenol red free RPMI was added, and images were acquired using a BZ-9000 fluorescence microscope (Keyence, Osaka, Japan) using a 10X PlanFluor NA 0.30 Ph1 lens with BZ filters for TRITC, GFP-B, and DAPI. Images were uniformly processed in Creative Cloud Photoshop CC using the brightness and contrast tools.

To stain cell organelles, early endosome and late endosome staining dye (Bacmam 2.0, Thermos fisher scientific), Lysotracker and NucBlue™ Live ReadyProbes™ reagents were used following the instruction noted in protocols provided by manufacturers. All the earlier mentioned nine samples were immobilized following the 4% paraformaldehyde fixation method. Finally, fixed and stained cells were imaged using a Nikon Eclipse Ti2 microscope (Nikon, Korea) coupled with 980 nm CW laser (EM595, Gooch & Housego).

In order to determine the effect of HLA-G and PD-L1 on interaction of immune cells with cancer cells, we co-cultured OVCAR-3 (stained using NucBlue Live ReadyProbes™, ThermoFisher Scientific) with PBMCs in 5:1 ratio in RPMI medium with or without HLA-G (3 ng/mL) and PD-L1 (3 ng/mL). We performed cinematography by taking pictures at interval of 30 s. The PBMCs were collection from human blood following Ficoll separation protocol (oral consent taken). In parallel, we used cells synchronized in G1 phase (lovastatin) and mitotic phase (nocodazole). The images were analyzed using Image-J. The cell was considered lysed as soon as cytoplasm started leaking and the immune cells were considered attached if their attachment remained constant for the 10 frames i.e., 5 min (Supplementary Video provided; Video S1; for unsynchronized cells in normal culture medium).

The preparation and imaging of tissue sections were performed as previously described by our lab (39 (link)–41 (link)). Briefly, tissue sections were fixed in 10% formalin and subsequently paraffin-embedded for H&E staining, which was performed by the University of Minnesota’s BioNet Histology Core. To measure superoxide production (oxidative stress) in AML12 cells, fluorescence imaging was performed using the superoxide indicator DHE (Abcam, catalog ab236206) and an ECHO fluorescence microscope and fluorescence plate reader. Briefly, live cells were incubated with 15 μM of DHE for 1.5 hours. NucBlue Live ReadyProbes (Thermo Fisher Scientific, catalog R37605) was added during the final 20 minutes to stain for nuclei. After staining with DHE and NucBlue, the cells were washed once, and stain-free media was added before imaging or reading on the plate reader.