Suggested Citation:
Demetri Spyropoulos, Lexi Temkin, Robert Bowers, John Baatz. 2016. Alligator Oil/Dispersant Exposure and Adipogenesis. Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/N7PG1PQH
Purpose:
To determine if oil/dispersant mixtures are obesogenic, i.e., drives differentiation of preadipocytes to an adipocyte phenotype, containing abundant lipid droplets.
Data Parameters and Units:
Adipogenic potential was measure using mean relative fluorescence intensity of the lipid specific marker AdipoRed as a function of oil/dispersant mixture concentrations of 0, 0.1, 1, 10 and 100 ppm. Rosiglitizone (1 uM) was used as a positive control. Alligator_Preadipocyte_OilDisp Exposure_Adipogenesis.xlsx: This file includes a graph and sample of the stained wells. Pixel count, black pixels indicating AdipoRed stained lipid accumulation (Adipogenesis) Concentrations of oil/dispersant mixture (100/10 ppm, 10/1 ppm, 1/0.1 ppm, 0 ppm) Rosiglitizone, positive control (1 uM Rosi) One well in each treatment for preadipocyte growth media, undifferentiated control (PGM) Average for the 3 trials, normalized (avg) Standard deviation for the 3 trials, normalized (stedev) Alligator Preadipocyte Oil_Dispersant Exposure and Addipogenesis Protocol.docx: This file contains a description of the methods used Alligator_Preadipocyte_OilDisp Exposure_Adipogenesis.tif, Alligator_Preadipocyte_OilDisp Exposure_Adipogenesis_225 Threshold.tif: Images of the stained wells Alligator_Preadipocyte_OilDisp Exposure_Adipogenesis_cell images.pdf: Images of stained cells Alligator_Preadipocyte_OilDispExposure_Aidpogenesis_graph.pdf: Copy of the graph included in file Alligator_Preadipocyte_OilDisp Exposure_Adipogenesis.xlsx Alligator_Preadipocyte_OilDisp Exposure_Adipogenesis_DIF.docx: Information about the dataset and dataset file list
Methods:
Alligator Preadipocyte Cell Culture Alligator iPSC derived preadipocytes, were plated onto 60 mm cell culture dishes coated with 0.1% gelatin (Millepore ES-006-B) supplemented with 10ug/mL hyaluronan (HA, Sigma #H7630-10MG) at 1 x 105 cells/dish. Cells were grown to subconfluency at hypoxic conditions (37oC, 1.5% O2, 5% CO2) in E2- media containing DMEM F12 media (Invitrogen 11039) supplemented with 10% HyClone FBS (Thermo Scientific SH30C70.03), non-essential amino acids (Invitrogen 11140-050), Glutamax (Invitrogen 35050-061), 50 U/mL penicillin, 50 µg/mL streptomycin, .125 µg/mL Fungizone® (Invitrogen Antibiotic/Antimycotic 15240) and Mycogone (Genlantis A200100). Cells were then harvested and plated for subsequent exposure experiments. Oil/Dispersant Treatment Exposure Cells were seeded at 5 x 10 4 cells/well onto 24 well gelatin coated tissue culture plates in E2- media (described above) and allowed to adhere overnight at normoxia (32oC, ambient O2, 5% CO2). The next day, cells were treated with indicated concentrations of oil/dispersant mixture. The oil/dispersant mixtures were prepared as follows. CoREXIT EC9500A dispersant, 20 µL, was added to a 1.5mL epitube, followed by 180 µL of MC252 source oil and mixed well. 20 µL of oil/dispersant was removed and added to an epitube containing 180uL of 18MΩ and again mixed well. Three more serial dilutions were similarly made by adding 20uL of previous oil/dispersant to 180uL of 18MΩ water. For treatment, 1.8mL of E2- media was aliquotted into each of four 2mL epitubes. Starting with the most dilute oil/dispersant treatment tube, we mixed the solution well, then added 2.1uL to one of the epitubes containing E2- media. This effectively resulted in a 0.1ppm solution to be applied to the cells. This was continued in ascending order creating 1, 10, and 100ppm treatments. Overnight E2- media was replaced with 400 µL new E2- media for the 0ppm treatment (2X) or with designated oil/dispersant treatment media in quadruplicates and placed in incubators for 48 hours. Adipogenic Differentiation After 48 hours of oil/dispersant treatment, all wells were replaced with preadipocyte growth media (PGM) containing MEM (Invitrogen 10370), 10% HyClone FBS (Thermo Scientific SH30C70.03), Glutamax (Invitrogen 35050-061), 50 U/mL penicillin , 50 µg/mL streptomycin, .125 µg/mL Fungizone® (Invitrogen Antibiotic/Antimycotic 15240) and were cultured until one day post confluence. The cell culture media was then switched to preadipocyte differentiation media (PDM) consisting of PGM supplemented with 31.25 nM dexamethasone (Sigma, D4902), 62.5 µM 3-isobutyl-1-methylxanthine (Sigma, I7018), 25 µM indomethacin (Sigma I7378) and .125 µg/mL insulin (Sigma, I9278) or PDM containing 1 µM rosiglitazone (Cayman Chemical, 71740) for a positive control. Differentiation media was added in triplicates, leaving one well for each treatment in PGM as an undifferentiated control. Cells were cultured for six days, with media changed every two days, then harvested and analyzed for lipid accumulation using AdipoRed staining. AdipoRed Staining and Analysis Cells were washed twice with PBS and fixed on ice in cold .45 µm filtered 4% paraformaldehyde in PBS for five minutes. Cells were then again washed twice with PBS and a final volume of 500 µL of PBS was added to each well. For staining, 15 µL of AdipoRed (Lonza, PT-7009) was added to each well and incubated in the dark at room temperature for 12 minutes. Cells were washed once with PBS and a final volume of 500 µL of PBS was added. Lipid accumulation was determined using pixel density measurements on high resolution, 10µm, Typhoon scanner images (excitation: blue 488, emission filter: 580 BP 30 Cy3, TAMRA, AlexaFluor546, voltage: 300PMT, sensitivity: normal). Image threshold was adjusted in Photoshop® to remove background, undifferentiated cell staining verified by visual inspection. Black pixels, indicating AdipoRed stained lipid accumulation, were then counted.
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