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  • br Lactate dehydrogenase assay LDH assay br Almost

    2020-08-07


    2.7.4. Lactate dehydrogenase assay (LDH) assay
    Almost 1 mL of MCF-7, HCT-116 and MG-63 PX 478 were grown of DME media in the presence of 10% FBS for 48 h. The cancer cells were washed with DMSO and treated with AgNPs and cAgNPs. The treated cells were kept as such for 24 h, and after this, the plate was put on the shaker to release the LDH enzymes by using LDH kit (Sigma Aldrich). The whole homogenized medium was transferred into the 
    microcentrifuge tube and centrifuge it for 10 min at 15000 rpm to re-move dirt and cell debris. The 100 μL of AgNPs and cAgNPs were put in the substrate and absorbance was measured by UV- spectrophotometer.
    2.7.5. Analysis of DNA fragmentation
    63 cells (1 × 106 cells/mL) treated with cAgNPs, were plated in six well plates with Dulbecco's Modified Eagle Medium contained 10% fetal bovine serum(FBS). These cancer cells were incubated under 5% CO2 for 24 h at 37 °C. After incubation, the whole medium was gently re-moved, and cells were washed with phosphate buffer saline, after this the medium free fresh serum was added and kept inside the CO2 in-cubator for 1 h at 37 °C.
    All the three cancer cells were starved for overnight and later wa-shed thoroughly with phosphate buffer saline (PBS). After that 1 mL of lysis buffer was added and mixed it properly and transfer it into a centrifuge tube. The whole mixture in the centrifuge tube was in-cubated at 37 °C for 1 h. After incubation, 5 μL of proteinase K added to the mixture and incubated for another 3 h at 50 °C. After the incubation again added phenol/chloroform and isopropyl alcohol in the ratio of 25:24:1. The two layers are formed the upper aqueous layer were gently transferred to the another Eppendorf tube, and absolute chilled ethanol was added to the tube and mixed thoroughly. The whole solution was centrifuged for 10 min at 13000 RPM. Gently supernatant (upper layer) was discarded, and the pellet was detached with a gentle shake and again treated with 70% ethanol to remove all impurities. The whole sample was again centrifuged for 5 min at 11500 rpm. After cen-trifugation, the supernatant was again discarded, and pellet was later dried at 37 °C for 2 h and resuspended by adding 50 μL of Tris EDTA (TE) buffer. The 10 μg/mL of DNA sample were later electrophoresed in 1% agarose gel contained ethidium bromide (EB) in TBE buffer gel tank for 1 h under 90 V. The gels were examined under UV transilluminator.
    3. Results and discussion
    The bacterial culture of Bacillus cereus (ATCC 14579) was utilized
    Fig. 2. FTIR Spectra of filtrate, AgNPs and cAgNPs synthesized from Bacillus cereus where (A) filtrate (B) AgNPs, (C) cAgNPs.
    Fig. 3. UV –spectrophotometer analysis showing peak at 412 nm and FTIR Spectra for cAgNPs after two months showing no changes in the functional group (Stability check).
    Fig. 4. EDX analysis of biosynthesized cAgNPs showing strong peak of silver confirms the presence at 3 KeV.
    Fig. 6. Thermogravemetric analysis showing % of protein loss (A) TGA of AgNPs showing 8.925% proteins loss (B) TGA of cAgNPs 10.910% protein loss at different temperatures.
    for the biosynthesis of AgNPs. The centrifuged bacterial culture (su-pernatant) was treated with 1 mM AgNO3, the color changes of the solution into pale yellow is due to the reduction of silver ions from Ag+ to Ag 0 [19,20]. The UV visible spectrophotometer showed absorption peak at lambda max 415 nm while for coated nanoparticles showed little higher lambda max at 420 nm, respectively; due to interband transition and surface plasmon resonance as shown as Fig. 1.
    The FTIR spectroscopy study showed the peak at 3262 cm−1 for NeH stretches of amides and also showed a peak at 1633 cm−1 for NeH bend of amines. Hence FTIR suggests the presence of amide and amine in the solution stabilizes and reduces the AgNPs. The cAgNPs also displayed the same functional group on the FTIR as shown in Fig. 2 [21,22].
    The stability of cAgNPs was analyzed by UV spectroscopy and FTIR analysis. The nanoparticle solution was kept in the refrigerator for two months. After that, the nanoparticles were analyzed for stability by UV 
    spectroscopy which showed the maximum absorption at wavelength 410 nm, while FTIR analysis showed the presence of amines and amides in the solution confirms the stability of nanoparticles (shown in Fig. 3).
    The exact elemental composition of AgNPs was revealed by EDX. The result showed the high peak of Ag confirms the presence of AgNPs and FESEM showed the morphology of the nanoparticles while data showed the composition of AgNPs (as shown in Fig. 4).
    The TEM analysis revealed the average shape, size, and dispersity of AgNPs and cAgNPs. The analysis showed AgNPs are uniformly dis-tributed, with spherical shape and the size ranges from 5.37 nm to 17.19 nm (Fig. 5). While, cAgNPs are little larger in size, spherical, and uniformly distributed but some nanoparticles agglomerate because of the strong interaction between AgNPs and BSA [23], the size of nano-particles ranges from 11.26 nm to 23.85 nm (as shown in Fig. 5).