The total Ag content was determined using AAS according to the above mentioned procedure. The results were normalized according to the cell number and expressed as % of the controls. Results are presented as mean standard deviation of 2 replicates. Cell viability Lactate dehydrogenase www.selleckchem.com/products/AG-014699.html assay The LDH assay is used to evaluate the degree of cellular membrane damage associated with leakage of the cyto solic LDH enzyme. The Cytotox Non Radioactive Cytotoxicity Assay Kit was used in a 96 well plate format. The cells were exposed to the AgNP dis persions at particle doses ranging from 5 to 100 ugmL in 100 uL for 4 and 24 h. After exposure, 50 uL of the supernatant was transferred to a new 96 well plate. The rest of the supernatant was discarded and the cells were lysed with 100 uL Triton 1% for 30 min at 37 C.
50 uL of the lysate was transferred to a new 96 well plate and 50 uL of reconstituted substrate was added to both the supernatant and the cell Inhibitors,Modulators,Libraries lysate plates. After 20 min incu bation at dark conditions, reactions in both plates were terminated using 50 uL stop solution. Absorbance was measured at 495 nm using a plate reader. The absorbance of the supernatant corresponds to the LDH release, whereas the sum of the absorbance of the supernatant and cell lysate corresponds to the maximum LDH release. The cell viability was calculated by dividing the LDH release to the maximum LDH re lease for each well. The control was Inhibitors,Modulators,Libraries set to 100% viability and the results were expressed as percentage cell viabil ity. The experiments were performed at least three times in triplicate wells for each time point and AgNP dose.
The interference of AgNPs with the LDH assay was tested in an acellular system, as well as by incubating cell lysates with AgNPs before performing the assay. The acellular interference was performed by Inhibitors,Modulators,Libraries incubating different concentrations of particles with reconstituted LDH substrate. The interference was found to be non significant. The interference of the AgNPs with the LDH assay in terms of possible enzyme inhibition was investi gated by incubating cell lysates with AgNPs for 0, 4 and 24 h before performing the LDH Inhibitors,Modulators,Libraries assay. Alamar Blue assay The AB assay is used to assess cell viability based on the reduction potential of metabolically active cells. BEAS 2B cells were seeded in transparent 96 well plates and ex posed to the AgNP dispersions at concentrations ran ging from 5 to 100 ugmL for 4 and 24 h.
After exposure, 10 uL of AlamarBlue reagent was added in each well Inhibitors,Modulators,Libraries and incubated for 2 h at 37 C. The fluorescence was measured at 560 nm excitation and 590 nm emission wavelengths using a plate reader. Results were expressed as percent http://www.selleckchem.com/products/BAY-73-4506.html age cell viability versus the control. The experiments were performed at least three times in triplicate wells for each time point and AgNP dose.