Millicell ez slide

THP-1 derived macrophages were seeded onto Millicell EZ slide (Millipore) 4 well glass slide and infected with LV-control vector, LV-let7g or LV-let7g-sponge. The macrophage monolayers were washed with PBS and fixed for 10 min in PBS containing 4% paraformaldehyde, permeabilized with PBS containing 0.1% Triton X-100 and then blocked with 5% BSA in PBS. Subsequently, cells were incubated with anti-bodies (1:100) against RelA, p50, RelB or p52 for 2 hr at room temperature, followed by incubation with the secondary anti-bodies conjugated with DyLight^TM 488-conjugated anti-rabbit or anti-Mouse IgG (H&L) for 1 hr at room temperature in the dark. Macrophage nuclei were labeled with 0.1 mg/ml DAPI (Molecular Probes, Carlsbad, CA) in PBS for 5 min at room temperature. Finally, the immunostained cells were mounted with PermaFluor (Thermo Scientific) on glass slides and the co-localization of RelA, p50, RelB and p52 with the nucleus analyzed by confocal laser microscopy (FV1000 IX-81; Olympus).

(A)For U2Os or 66cl4 cell lines: Cells were seeded at a density of 80,000 cell per well of four-well glass Millicell EZ-Slide (Millipore, PEZGS0416) overnight in complete growth media, DMEM or RPMI respectively. 24 hr post-seeding, cells were starved in media containing 0.5% FBS + 1% Pen/Strep. 16 hr later, media was replaced with either DMEM/RPMI containing a total of 2 g/L glucose as follows: for U2Os cells low-glucose DMEM + 1 g/L D-(+)-Glucose + 10% FBS + 1% Pen/Strep was used; and for 66cl4 RPMI + 1 g/L D-(+)-Glucose + 10% FBS + 1% Pen/Strep. Cells were incubated for 12 hr at 37°C 5% CO2 and then prepared for immunofluorescence staining. (B) For MM6, cells were grown overnight at 1 million cells/ml in RPMI containing 10% FBS and 1% Pen/Strep. On the next day, cells were washed twice with 1x PBS (Wisent Biologicals, Cat# 311–010 CL) and resuspended in RPMI containing a total of 2 g/l glucose + 10% FBS + 1% Pen/Strep and incubated at 37°C 5% CO2 for 4 hr after which prepared for fluorescence staining. CD34+ cells were purchased from ATCC and primary high-eIF4E AML samples were obtained from our phase I clinical trial of ribavirin and low-dose cytarabine (Assouline et al., 2009 (link); 2015 (link)).

Cells were plated in 8 compartment plates Millicell EZSlide (Millipore, Massachusetts, USA) at 1x10⁵ cells per well. After treatments, EZ slides were incubated with AR primary antibody (ab9474) overnight. Then, secondary antibody coupled to ALEXA fluorophore was added to the plate. Three different fields at 40 X were taken for each treatment with a confocal microscope.

INS-1 cells were seeded into Millipore’s Millicell EZ SLIDE and incubated overnight. Next, the cells were treated with different drugs and culture for 24 hours. Then, TUNEL staining kit was used to conduct apoptosis analysis according to the manufacturer’s instructions. Finally, TUNEL-positive cells were observed under a fluorescence microscope. TUNEL positive cells were quantified and expressed as percentage of total cells.

Melanoma cells were seeded on Millicell EZ slide (PEZGS0416, Millipore, Burlington, MA, USA) and after 12 h were fixed in 4% formaldehyde (FA) for 10 min at RT. Subsequently the cells were permeabilized with 0.1% Triton X−100 for 10 min at RT and then blocked with 2% BSA in PBS for 1h. Slides were incubated for 1h at RT with primary antibodies directed against ENO1 (dilution 1:50). The slides were then washed in PBS and incubated for 45 min with secondary anti-Goat IgG antibodies conjugated to fluorescein isothiocyanate (FITC) (554020, Becton Dickinson, Franklin Lakes, NJ, USA) and DAPI. The slides were washed in PBS and mounted with polyvinyl alcohol mounting medium with DABCO (10981 Sigma–Aldrich, St. Louis, MO, USA).
A Stellaris 8 laser confocal scanning microscope equipped with 63x NA1.4 oil objective (Leica) was used to image samples. All images were taken at the same settings and further analyzed using FIJI software [22 (link)]. Images were filtered to remove noise (Median filter, radius = 2), then a triangle threshold was applied to segment cells from which mean fluorescence signals were measured. Data were exported to Excel software and analyzed with a t-test.