Thiazolyl blue tetrazolium bromide mtt reagent

Cells were plated in a 96-well plate before being treated daily with media only, or media containing doxycycline and/or GRP78 N-20 antibody or IgG control. The cells were next treated with cisplatin at the concentrations and times indicated. Following incubation, the media was aspirated from the cells and replaced with 50uL of fresh media containing 0.5 mg/mL Thiazolyl Blue Tetrazolium Bromide (MTT) reagent (Sigma) per well. The cells were incubated for 4 hours at 37 degrees. After incubation, 150 uL of DMSO was added to each well and mixed to completely dissolve the solution. Absorbance was measured in plate reader at 570 nm.

Anti-c-Src MAb-327 [84 (link)], provided by J.S. Brugge, Harvard University. Anti-Fak, anti-Cyr61, and anti-cyclin D1 were from Santa Cruz Biotechnology. Anti-CD63 (Inmuno-Step; Calbiochem). Antibodies to MMP2, MMP9, and MAb 4G10 were from Merk-Millipore. Anti-MMP7 was from Abgent. Anti-pY397-Fak, secondary horseradish peroxidase-conjugated antibodies, siRNA-hs-Cyr61 (s7244, Silencer^® selected and validated siRNA), siRNA-hs-c-Src (s13414, Silencer^® selected and validated siRNA), and scramble siRNA (Stealth RNAi Negative Control Duplex #12935–300) were from Life Technologies. Anti-pY925-Fak was from Cell Signaling Technologies. Anti-paxillin, anti-pY118-paxillin, anti-p130CAS, anti-caveolin-1, anti-pY14-caveolin-1, anti-p27^Kip1, and Matrigel^TM were from BD-Biosciences. Anti-α-tubulin, anti-ß-actin, doxycycline (Doxy), Trypan blue, Thiazolyl Blue Tetrazolium Bromide (MTT reagent), and esiRNA human Rab27a were from Sigma-Aldrich. Anti-Rab27a polyclonal antibody (Peter van der Sluijs, University Utrecht, [85 (link)]). Anti-Tet-repressor (Mobitec). Blasticidin and zeocin were from InvivoGen. Tet-Free-FCS (PAA Laboratories GmbH). Acrylamide/Bis-acrylamide (29:1), SDS and ammonium persulfate were from Bio-Rad Laboratories. ECL was from GE Healthcare Biosciences. BCA protein assay and DharmaFECT 4 were from Thermo Scientific.

Ciprofloxacin powder (CIP; Mw = 331.35; purity > 98%) was donated by Teva Pharmaceutical Works Ltd. (Debrecen, Hungary) for research work. Polyvinylpyrrolidone (PVP; Mw = 1,300,000) was purchased from Alfa Aesar (Heysham, UK). The solvents for the ES solutions, the ethanol (99.99% purity), and the glacial acetic acid were obtained from Fisher Scientific (Loughborough, UK) and Sigma-Aldrich (Hamburg, Germany), respectively. As a reference for the in vitro dissolution studies, commercially available CIP-containing filmcoated per os tablets (Ciprinol^® 250 mg, KRKA, d. d., Novo mesto, Slovenia) were used. Acetonitrile was purchased from Molar Chemicals (Halasztelek, Hungary). The thiazolyl blue tetrazolium bromide (MTT) reagent and sodium dodecyl sulfate were obtained from Sigma-Aldrich (Hamburg, Germany). Phosphate buffer solutions (PBS; pH 2.8 and pH 7.4) were prepared in-house. All other chemicals were analytical grade, and purified water was used.

Dopamine hydrochloride, NH₄OH (28–30%), PBS (0.01 M, pH 7.4), N-hydroxysuccinimide (NHS), N-(3-Dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC), PTX (from semisynthetic, >97%), Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS, USA origin), thiazolyl blue tetrazolium bromide (MTT reagent) and dimethyl sulfoxide (DMSO) were all supplied by Sigma–Aldrich (Darmstadt, Germany). Penicillin-streptomycin (5000 U/mL), calcein AM, propidium iodide ReadyProbes^TM reagent, Gibco^TM Trypsin-EDTA (0.25%) and trypan blue stain (0.4%) were obtained from Thermo Fisher Scientific (Eugene, OR, USA). 2-PrOH and HCl (37%) were purchased from PanReac Química S.L.U. (Castellar del Vallès, Barcelona, Spain). Tmab was gifted from the Instituto de Biología Molecular y Celular del Cáncer (Salamanca, Spain) and the FITC Annexin V apoptosis detection kit with 7AAD was obtained from Immunostep (Salamanca, Spain).

Cells were seeded in 96-well plates at optimized
densities of 1 × 10³ cells per well (DU145, PC-3),
6 × 10³ cells per well (LNCaP), 2 × 10³ cells per well (PWR-1E), or 4.5 × 10³ cells per
well (RWPE-1). Cells were allowed to adhere for 24 h before drug treatments
were applied. For serum deprivation, treatments were applied in a
serum-free medium. For antagonist experiments, cells were pretreated
with antagonists for 1 h before the addition of CBD. After drug treatment,
5 mg/mL thiazolyl blue tetrazolium bromide (MTT) reagent (Sigma-Aldrich)
was added to each well, and the plates were incubated at 37 °C.
After 3 h of incubation, the culture medium and MTT reagent were discarded,
and the formazan crystals were dissolved using DMSO. Absorbance was
measured at 570 nm using a CLARIOstar microplate reader (BMG Labtech).
The percentage viability was calculated relative to the vehicle control.