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Pharmaceuticals 2020, 13, 137 7 of 9 4.2. Cell Culture Human adenocarcinoma cells (A549) were purchased from ATCC (ATCC, Manassas, VA, USA). The cells were cultured in Ham’s F-12K Nutrient Mixture, Kaighn’s Mod. All medium was supplemented with 10% fetal bovine serum (FBS) and 1% penicillin streptomycin. Cells were grown in a humidified incubator at 37 ◦C with 5% CO2. For plating, cells were washed with phosphate-buffered saline (pH 7.4), trypsinized using Ethylenediaminetetraacetic acid (EDTA), and counted in a hemocytometer using 0.4% trypan blue. 4.3. Phantom Construction Tissue-mimicking phantom construction was previously described by our group [13]. Briefly, gel phantoms were constructed using a combination of intralipid as the scattering agent, India ink as the absorber, and agar powder to make the substrate. The intralipid and India ink concentrations were chosen to produce phantoms with reduced scattering coefficient (μs’) of 7 cm−1 and an absorption coefficient (μa) of 0.26 cm−1 at 630 nm, and μs’ of 7.05 cm−1 and μa of 0.24 cm−1 at 532 nm [13]. The phantoms were 3- or 5-mm thick. 4.4. In Vitro PDT Cells were plated in wells at a seeding density of 2500 cells in 100 μL of media. After incubation for 24 h, the media was replaced with media containing TLD1433 to give final concentrations of 0.01 μM to 60 μM. Following 60 min incubation, the media (containing any TLD1433 not taken up by cells) was replaced with fresh media without TLD1433. Within 3-5 min, the TLD1433-treated cells were exposed to the therapeutic light. This procedure of TLD1433 media replacement follows previous methods described by Kaspler et al. [9]. The cells were illuminated with either a 630-nm or 532-nm light. In one set of experiments, the light was delivered through a laser fiber with a micro-lens (front diffuser, FD1, Medlight SA, Ecublens, Switzerland). A 630-nm diode laser (ML-6500-630, Modulight Inc., Tampere, Finland) was used to illuminate the plate with an irradiance of 65 mW/cm2 and fluence of 230 J/cm2 with a spot size of 3-cm. A 2-W fiber coupled 532-nm diode laser (LSR532H-2W-FC, CivilLaser, Hangzhou, Zhejiang, China) was used to deliver 20 J/cm2 with an irradiance of 28 mW/cm2 with a spot size of 5-cm. After light treatment, the plates were returned to the incubator for 24 h. In another set of experiments, the cells were illuminated with the OSA, using two 2-cm radial diffusers (RD20, Medlight SA, Ecublens, Switzerland) that were placed 3 cm apart in parallel channels (Figure 3A). Each fiber transmitted 532-nm light at 50 mW/cm for 278 s (a total 200 mW/cm for 55.6 J/cm). Plates were either placed in direct contact with the OSA or on top of a 3-mm to 5-mm phantom (Figure 3B–D). This set up mimics the configuration where the OSA light is delivered in one direction towards the pleural in the chest cavity (Figure 3B,C). In another configuration, the OSA was placed on a phantom that acted as a backscattering layer (Figure 3D) that could be present due to surrounding tissue as we previously reported [13]. All PDT-treated cells were evaluated alongside controls, including cells treated with light but not TLD1433 and cells treated with TLD1433 but not light. Controls for assessing cell growth in the absence of TLD1433 or light were also included. All experiments were conducted in triplicate. After light treatment, the plates were returned to the incubator (the 37 ◦C, 5% CO2) for 24 h. Cell viability was then measured with a resazurin cell viability assay. Resazurin was incubated (37 ◦C, 5% CO2) with the cells for 4 h, then measured by quantifying fluorescence (excitation at 570 nm, emission at 585 nm) using a Varian, Cary Eclipse Fluorescence Spectrophotometer plate reader. The cell viability was normalized to growth control. Results of PDT response were plotted using Graphpad Prism, while viability maps were generated using SigmaPlot.PDF Image | TLD1433-Mediated Photodynamic Therapy Lung Cancer Cells
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