Abstract:
Alligator induced Pluripotent Stem Cells (iPSCs) were treated with various concentrations of oil/dispersant mixture (MC252 source oil/dioctylsodium sulfosuccinate [DOSS], at constant ratio of MC252:Doss of 1:10) to assess viability of cells after 48 hour exposure. Viable cells were labeled with sulforhodamine B (SRB) and absorbance was quantitated using the Typhoon scanner. There was a trend toward increasing cell viability with increasing concentrations, with maximum viability observed at 100ppm oil/dispersant.
Suggested Citation:
Demetri D Spyropoulos, Lexi Temkin, Robert Bowers, John E Baatz. 2017. Alligator induced Pluripotent Stem Cell Oil/Dispersant Toxicity Test. Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/N73F4N06
Data Parameters and Units:
Viability was measured using average mean relative fluorescence intensity (Relative Light Units; RLU) of sulforhodamine B after exposure of alligator iPSCs to 0, 0.1, 1, 10, 100 and 1000 ppm Dioctyl sodium succinate (DOSS).
Methods:
General Alligator culturing conditions Alligator iPSCs were maintained on 2% Matrigel (BD Sciences #354234) coated 60mm plates. All cells were expanded in mTeSR1 basal medium (Stem Cell Technologies # 05871) containing mTeSR Supplement (Stem Cell Technologies #05852), Glutamax (Invitrogen #35050-061) and Mycogone (Genlantis #A200100), all at manufacturer specified concentrations and at 37°C in hypoxia (1.5% O2, 5% CO2). When plates reached near-confluence, cells were trypsinized and counted using a hemocytometer. Toxicity Testing Alligator iPSCs were plated on gelatin-coated 24 well culture plates at a density of 1x104 cells/well in 0.3mL of mTeSR media specified above. Cells were allowed to attach to plates overnight at 37°C in normoxia (ambient O2, 5% CO2). The following morning, 20uL of COREXIT® EC9500A was mixed with 180uL of MC252 source oil by pipetting up and down 20 times. 20uL of oil/dispersant was removed and added to an epitube containing 180uL of 18MΩ water. Again, the solution was mixed thoroughly by pipetting up and down at least 20 times. Three more dilutions were similarly made by adding 20uL of oil/dispersant to 180uL of 18MΩ water. 1.8mL of mTeSR media was aliquotted into each of five 2mL epitubes. Starting with the most dilute oil/dispersant treatment tube, the solution was mixed well, then 2.1uL was added to one of the epitubes containing mTeSR media. This effectively resulted in a 0.1ppm oil/dispersant solution to be applied to the cells. We continued in ascending order to the most concentrated oil/dispersant treatment tube, creating 1, 10, 100 and 1000ppm treatments. Media was aspirated from the four 0ppm treatment (no treatment control) wells and 400uL of fresh mTeSR media was added. mTeSR media was aspirated from all other wells, one row at a time, and 400uL of the appropriate treatment media was added generating quadruplicates for each concentration oil/dispersant. Plates were swirled and put at 37°C in normoxia for 48 hours. SRB labeling cultured cells After the 48 hour treatment plates were removed from the incubator and 100uL of cold 5% trichloroacetic acid (TCA) was added to each well containing 400uL of media. Plates were incubated for one hour at 4°C, and swirled once at 30 minutes. Plates were washed by filling an ice bucket with cold tap water and submerging the plate right side up, lowering it under the surface of the water. Plates were inverted to dump out the wash. This procedure was repeated three times for a total of four washes. Plates were tapped upside down on paper towels to drain, then inverted and left to air dry at room temperature before staining. Once plates were dry 200ul of 0.4% SRB (200mg dissolved in 50mL 1% acetic acid) was added to three wells per treatment (one well was left unlabeled as a background control) and plates were incubated for 30 minutes at room temperature in the dark. SRB was aspirated and wells were washed two times with 250uL of 1% acetic acid and two times with 500uL of 1% acetic acid, aspirating between washes. Plates were inverted and left to air dry in the dark at room temperature. Once plates were dry, 400ul of 10mM tris base was added to each well and plates were swirled to promote even distribution of SRB stain. Creating the SRB dilution plate 400uL of 10mM tris base was added to each well of a 24-well cell culture plate. 50mL of 0.4% Sulforhodamine B (SRB, Sigma #S1402-5G) was prepared by dissolving 200mg of SRB powder in 50mL of 1% acetic acid. 400uL of 0.4% SRB was added to the first well of the culture plate and mixed well. 400uL was removed from this well and added to the next well. We continued making 11 two-fold serial dilutions of SRB (1722nM to 1.68nM SRB), leaving a twelfth well containing tris base only. We repeated this procedure to create a duplicate of the dilution series. The plate was sealed with parafilm and stored in the dark at room temperature for up to one month. Typhoon scanning and data analysis Treated plates and the SRB dilution plate were scanned together on the Typhoon Scanner. It was important to scan both plates at the same time, as the dilution plate acts to normalize the Typhoon so that the comparison of scans made on different days is possible. Typhoon scanner settings were as follows: 24well fluorescence, Emmax = 580nm, 200um pixel size, +3mm focal plane, 580 BP, 320 PMT, Green (532) laser (Dry Scan). Once plates were scanned, data were quantitated using ImageQuant software and exported to Excel for analysis. Absorbance values from the SRB-labeled wells were normalized to the background control well for that treatment and average absorbance and standard deviations were calculated.
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