Gel documentation system

Fragmentation of DNA into oligonucleosomal bands, as a function of apoptotic cell death, was studied by DNA laddering assay as described before [14 (link)]. Apoptotic DNAs from 1 ml of KalsomeTM10 treated and untreated L. donovani promastigotes (1x10⁷/ml)) were isolated using Suicide Track^™ DNA Ladder Isolation Kit (Calbiochem, Germany). Briefly, the treated and untreated cells were suspended in 500 μl of extraction buffer which separates apoptotic DNA from high molecular weight DNA. The cells were then incubated on ice for 30 min and centrifuged at 15000 x g for 5 min. Supernatants carefully removed and transferred to a clean tube. RNA in the supernatant was then degraded using solution 2 of the kit. Subsequently, DNA from the cell lysate was isolated using solution 3 and incubation at 50°C for 1 h. The DNA pellet obtained after centrifugation was washed with 70% ethanol followed by 100% ethanol. It was further air dried and resuspended in buffer (10 mM Tris pH 7.5, 1 mM EDTA). DNA aliquots were electrophoresed on 1.5% agarose gel containing ethidium bromide (0.5μg/ml), using Tris—acetate—EDTA (pH 8.0) running buffer, and then observed and photographed under UV illumination in a BioRad gel documentation system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Gel photography and documentation were carried out using the Bio-Rad gel documentation system. The number of bands revealed on each gel lane was counted and compared using the Gel Pro-Analyzer software. Quantitative variations in band number and concentration were estimated using the Bio-Rad video densitometer, Model Gel Doc 2000. With regard to variation in protein banding patterns, electropherograms of each exposed and nonexposed sample were scored for the presence or absence of bands.

PCR amplified products were analyzed by agarose gel electrophoresis as follows: 1.5% agarose gel (cat. # CA3510-8, Denville Scientific, Inc.). Gel was prepared in 1× Tris-borate-EDTA buffer (cat. # 28355, Thermo Fisher Scientific). SYBR Safe (cat. # S33102, Invitrogen) was added to the agarose gel before polymerization. Agarose gel was loaded with 5.0 µl of each PCR amplicon product mixed with 6× loading dye (cat. # R0611, Crystalgen) and ran for 20–25 min at 200 V. Each agarose gel was loaded with standard 100 base pair markers/ladder (cat. # 65-0321, Crystalgen). The gel was exposed and captured for imaging using a Bio-Rad gel documentation system (Bio-Rad, Hercules, California). Intensity/densitometry of agarose gel band was measured by Image J.

After treated the cells with different concentrations of DEN or DEN-HPβCD, Total RNA was isolated by using a Trizol RNA isolation kit (Invitrogen, Carlsbad, CA, USA) and stored at −80 °C for subsequent analysis. First-strand cDNA was synthesized from 600 ng of RNA by using iScript cDNA Synthesis kit (Bio-Rad). MyTaq Mix kit (Bioline, Taunton, MA, USA) was used for amplification. The sequences of primers used in RT-PCR were shown in Table 1. The PCR cycling conditions for caspase-3 were 95 °C for 1 min and the amplification was followed by 30 cycles of denaturing at 95 °C for 15 s, annealing at 60 °C for 15 s, and primer extension a 72 °C for 10 s with a final extension at 72 °C for 7 min. The PCR cycling conditions for β-actin were 95 °C for 1min and the amplification was followed by 30 cycles of denaturing at 95 °C for 15 s, annealing at 61 °C for 15 s, and primer extension a 72 °C for 10 s with a final extension at 72 °C for 7 min. The PCR product was analysed on 1.5% agarose gels and DNA bands were visualized by 5× loading buffer and Bio rad gel documentation system/Bio-Rad. All experiments were performed in triplicate.

Protein expression level was analysed as previously described [3 (link)]. Protein lysates were separated on a 10% SDS-PAGE and protein bands were semi-dry transferred onto PVDF membranes. Membranes were blocked by 5% BSA before the incubation of primary antibodies. All primary antibodies were diluted using 5% BSA in TBST buffer and incubated overnight at 4 °C except pErk1/2 antibody and GAPDH, which were incubated for 1 h at room temperature. GAPDH was used as the loading control. Then, membranes were incubated with secondary antibody (HRP-anti-rabbit IgG) at room temperature for 1 h. Blots were imaged using enhanced chemiluminescence detection on the Bio-Rad gel documentation system (BioRad, Sydney Australia), and densitometry analysis was performed using Image Lab 5.2.1 (BioRad, Sydney Australia).

TcdA (1 μg) (List Biological Laboratories, Inc. California, USA) was incubated with DMC-4 (35 μl) for 1 h at 37 °C. Samples were loaded using 6X SDS loading buffer and heated at 95 °C for 10 min. All samples were loaded on to gradient SDS-PAGE gels (4-15%) after boiling. Silver staining was performed using the Pierce Silver Stain Kit as per the manufacturer’s instructions (Thermo Fisher Scientific. USA).
TcdA and TcdB (100 ng) were incubated with DMC-4 (35 μL) for 1 h at 37 °C. Samples were loaded using 6X SDS loading buffer and heated at 95 °C for 10 min. All samples were loaded into gradient SDS-PAGE gels (4-15%) after boiling. The polyvinylidene difluoride membrane (EMD Millipore, Germany) was incubated with either TcdA mouse monoclonal antibody (1:2500, Santa Cruz Biotechnology, Inc., USA, PCG4) or TcdB sheep polyclonal antibody (1:1,000 dilution, R&D Systems, USA, AF6246) overnight at 4 °C. After, the membrane was incubated with secondary antibody (1:20,000 dilution, goat anti-mouse HRP, Invitrogen, USA, CAT: 31430 for TcdA and 1:10,000, Donkey anti-Sheep HRP, Sigma-Aldrich, MO, USA, CAT: A3415 for TcdB) for 1 h at room temperature. Imaging of the membrane was performed using a Bio-Rad gel documentation system equipped with the Image Lab software^{26 (link)} (BioRad Laboratories Ltd., Canada).