Palmitoleic Acid and Algae Extract C ollaboration

1. Palmitoleic Acid and Algae Extract Collaboration S. Vanessa Aguilar Dr. Christine Schmidt Dr. Rhykka Connelly 11/16/2011

2. Yesterday we won intramural championship

3. GOALS 1 Kato, et al, Nutrition and cancer, 2007. 2 Yang, et al, Lipids in health and disease, 2011.

4. Background Using fatty acid-rich albumin in human embryonic stem cells (albuMAX) triggers adipogenic features. Ruiz-Vela et al., Stem Cell Rev and Rep 2011.

5. Aims

6. Aim 1

7. Background • Cell viability assay for fatty acid treatment • Vehicle • Dimethylsulfoxide (DMSO)1 • Ethanol 2 • Concentration • 10 μM1 • 125 μM1 Ruiz-Vela et al., Stem Cell Rev and Rep, 2011 Kato et al, Nutrition and Cancer, 2007

8. Methods Ethanol vehicle DMSO vehicle μM μM Control Control max max 250 25 25 2.5 2.5 250 • Seed cells 5,000 cells/well • Test palmitoleic acid concentration in ethanol and dimethylsulfoxide (DMSO). • Check for proliferation using CellTiter 96 kit (Promega) 1, 2 and 3 days with tested media Control

9. Palmitoleic Acid in DMSO • DMSO itself is killing the cells 5% DMSO • 250 μM can’t be tested using DMSO • 2.5 μMand 25 μMof palmitoleic acid is safe

10. 3 Days Palmitoleic Acid in DMSO Control DMSO (highest 5%) 25 PA μM in DMSO 2.5 PA μM in DMSO 250 PA μM in DMSO

11. Palmitoleic Acid in Ethanol • 2.5 μM and 25 μM of palmitoleic acid is safe • 250 μM of palmitoleic acid cells are not proliferating and dying

12. 3 Days Palmitoleic acid in Ethanol Control Ethanol (highest 5%) 25 PA μM in Ethanol 2.5 PA μM in Ethanol 250 PA μM in Ethanol

13. Cell Morphology 250 PA μM in Ethanol 3 days 10x 250 PA μM in Ethanol 3 days 20x

14. Cell Viability 2.5 uM and 25 uM of PA in both Ethanol and DMSO are safe to use with fibroblast in vitro

15. Conclusions • DMSO is not a good vehicle for palmitoleic acid. • Ethanol is a good vehicle for lipids. • There is a cell response with 250 μMpalmitoleic acid.

16. Aim 2

17. Test algae extract • Algae extract was dried and weighed (58.7 mg) • Dr. Connelly ran a gas solid chromatography to identify the lipid soluble components. • Results • Fatty acids 2.864 mg • Myristic acid – 248 μg (8.6%) • Palmitic acid – 415.5 μg (14.5%) • Oleic acid – 85 μg (3%) • Palmitoleic acid – 1344 μg(47%) • Linoleic acid -200 μg (7%) • Linolenic acid- 96 μg (3.3% • Eicosapentaenoic acid -476 μg (16.6%) • Other 55.83 mg • Carotenoids/xanthophills • Vitamins • Anything lipid soluble

18. Methods Algae Extract Palmitoleic Acid Ethanol vehicle μM μM Control Control max max 250 100 100 25 25 250 • Seed cells 2,500 cells/well • Test palmitoleic acid concentration in ethanol and algae extract. • Check for proliferation using CellTiter 96 kit (Promega) 1, 3 and 7 days with tested media. Control 2 Control 1

19. Palmitoleic acid

20. Algae Extract

21. Cell Viability

22. Conclusions • Day 3 did not show increase of proliferation. • Checking on the images I took after adding MTS assay, my MTS assay was killing the cells. • Day 7 there are no differences between controls, 25 μM, and 100 μM of Palmitoleic acid and 25 μM of algae extract.

23. Aim 3

24. Methods • After Cell titer 96 • Fixed with 4% PFA at room temperature for 30°C minutes • Rinse 3x5 in PBS • Stained with LipidTox red Neutral stain (Invitrogen) 1:200 in PBS following Invitrogen protocol

25. Controls Day 1 Control Cell Titer and Lipid Tox Control LipidTox only

26. Palmitoleic Acid Day 1 Ethanol control 25 μMPalmitoleic acid 250 μMPalmitoleic acid 100 μMPalmitoleic acid

27. Algae Extract Day 1 25 μM Algae extract 100 μM Algae extract 250 μM Algae extract

28. Conclusions • Qualitatively, there is an increase in lipids inside cells. • Check pH of the testing media.