Lab tek 2 chamber slide

The phototoxic effect of AvIR was assessed by using the LIVE/DEAD Cell Imaging Kit (Thermo Fisher Scientific). Cells were seeded onto an 8-well Lab-Tek II chamber slide (Thermo Fisher Scientific) at a density of 10,000 cells/well 1 day before AvIR-PIT. The next day, the cells were treated by adding biotinylated anti-CEA (Bio-CEA), biotinylated anti-EpCAM (Bio-EpCAM) (5 µg/ml), or an equal volume of DPBS for 30 min, followed by adding AvIR (5 µg/ml) with another incubation for 30 min. The cells were exposed to NIR light (3 J/cm²) from a light-emitting diode (LED) light source (Shiokaze Giken, Niigata, Japan), which emits red light with a peak at 690 nm. The irradiation energy density was measured with a PM100D optical power meter (Thorlabs, Tokyo, Japan). The irradiated cells were incubated with a mixture of Live Green and Dead Red solutions for 20 min and were subsequently imaged using a fluorescence microscope BZ-9000 (Keyence, Osaka, Japan). To assess the target specificity, AvIR-mediated PIT was also performed for co-cultured CHO-CEA and CHO-EpCAM cells. The CHO-CEA cells were stained with CellTracker Blue dye (Thermo Fisher Scientific) and were co-cultured with unlabeled CHO-EpCAM cells in a Lab-Tek II chamber on the day before AvIR-PIT.

Experimental data was obtained from Schreiber et al. (2010) (link). To determine the neurological effects of PBDEs, primary human fetal neural progenitor cells (hNPCs) were cultivated in the form of neurospheres and exposed to BDE-47. Neurospheres were exposed to BDE-47 over a period of 7 days, with half the medium (with a chemical concentration of 1 μM) being refreshed every second day. Exposure occurs in the Lab-Tek II Chamber slide (Thermo Fisher Scientific), which has a flat, square-based format. Culture area is reported at 0.7 cm²/well, with a total well volume of 907 μl and a working volume of 500 μl.
The medium used in these experiments was a mixture of Dulbecco’s modified Eagle medium (DMEM) and Ham’s F12 (3:1) with no supplementation with additional serum. Fischer et al. (2017) (link) determined the lipid and protein concentrations within the DMEM solution as approximately 0.2 ml/L and 0.75 (0.69–0.86) ml/L respectively. The bulk medium in the model simulations was therefore parameterized to match these volume fractions. Again, we simulated the potential influence of other dissolved organics on the distribution of the chemical following the same approach as described in Case study 1. The partitioning properties compiled for BDE-47 are summarized in Table 1.

A Lab-Tek® II Chamber slide (Thermo Fisher Scientific, Waltham, MA) was
pre-coated with a 70 μl Matrigel matrix and polymerization was allowed for in a
37°C incubator for 10 to 15 minutes. Cells in the growth medium mixed with the
indicated concentration 0 or 10 μM of quercetin and 2% Matrigel matrix were
transferred onto the chamber slide. Once the gel was polymerized, cells were
allowed to grow with the changing media every other day for 6 days. Colony
formation was photographed under 200× magnification.

Cells were plated at 30,000 cells per well in 8-well Lab-Tek II chamber slide (Thermo Fisher Scientific) 24 h prior to transduction. 24 h after plating, cells were transduced with AAVHSC7-226 at a MOI: 150,000. Before staining, cells were washed with PBS and fixed with 4% paraformaldehyde 48 h after transduction. They were then rinsed 3-times with PBS and blocked with PBS containing 3% BSA and 0.3% Triton X-100 for 1 h at room temperature. This was followed by incubation with primary anti-MeCP2 antibody (1: 100 dilution; Cell Signaling, #3456) and an anti-GFP antibody (1:500 dilution, Thermo Fisher, # MA5-15256) in PBS containing 1% BSA and 0.3% Triton X-100 at 4°C overnight. The cells were then rinsed 3-times with PBS and incubated with secondary antibodies. Secondary antibodies used were anti-rabbit Alexa Fluor-555 IgG (1:500, cell Signaling, #4413) and anti-mouse Alexa Fluor-488 IgG (1:500, cell Signaling, #4408) in PBS containing 1% BSA and 0.3% Triton X-100 for 2 h at room temperature in dark. The cells were finally rinsed 3-times with PBS, mounted with antifade reagent containing DAPI (VECTASHIELD Vibrance^®, #H-1800) and visualized at ×40 magnification using the Zeiss LSM 700 confocal microscope.

THP1 Macrophages and NS-SV-TTAC Acinar Cells were plated on an 8-well Lab-Tek II Chamber Slide (Thermo Scientific, 154534). After indicated treatment, cells were loaded with Quant-iT PicoGreen Reagent (1:50) (Invitrogen, #P7589A) and MitoTracker Red CMXRos (Invitrogen, #M7512) (1:10000) and incubated for 45 minutes at 37°C. The cells were fixed for 20 minutes at room temperature in 4% PFA. The cells were blocked in 3% BSA diluted in 1X PBS for 1 hour. Cells were washed with 1X PBS and then stained with primary antibodies overnight and then stained by secondary antibodies for 1 hour at room temperature (Supplemental Table 2). Images were acquired on Nikon A1 HD (Nikon) confocal microscope and processed with CellProfiler in ImageJ (Broad Institute).

MCF10A cells were cultured in DMEM/F12 (Invitrogen, Carlsbad, California) supplemented with 5% horse serum, 20 ng/ml EGF, 0.5 µg/ml hydrocortisone, 10 µg/ml insulin, 100 ng/ml cholera toxin, and 50 µg/ml penicillin/streptomycin. Cells were maintained in a 37°C incubator at 5% CO₂. Cells were cultured on Lab-Tek II Chamber Slide (Thermo Scientific, Waltham, Massachusetts) to 80–85% confluent, and then siRNA transfections were performed twice using Lipofectamine RNAiMAX Reagent with a 24-hr interval. Cells were processed for immunoblotting or immunofluorescence 96 hr post-transfection. SMARTpool siRNA oligonucleotides toward human SPTAN1 and Non-Targeting siRNA Pool #2 (control siRNA) were purchased from GE Dharmacon (Lafayette, Colorado). For immunostaining, cells were fixed and stained following standard method using anti-YAP antibody (Novus, 1:300 dilution) and anti-p-MLC antibody (1:10, Cell Signaling Technologies, Beverly, Massachusetts). Western blotting was done using antibodies against following proteins: pYAP127 (1:1000, Cell Signaling Technologies); pYAP381 (1:1000, Cell Signaling Technologies); YAP (1:200, Sigma, St. Louis, Missouri); MLC (1:1000, Cell Signaling Technologies); SPTAN1 (1:500, Santa Cruz Biotechnology, Dallas, Texas); and beta-actin (1:200, Sigma).