Astaxanthin and Cellucor Poster Fall 2015
1
Cellucor C4 • Learn what ingredients in Cellucor caused the MTT assay to react in blank wells. • Try another type of assay to avoid the MTT reaction. • Test using different cell lines. Astaxanthin • Vary the range of dilution. • Use solvent such as DMSO to allow use of the whole supplement. • Find alternate forms of sterilization. What CHO Cells Say About the Supplements, Cellucor C4 and Astaxanthin Brian Eccleston, Bipina Acharya, Ciara Boatright, Jordan Cox, Brandon Curry, Kameron Killam, Blake King, Samantha Rice, Jennifer Yang, Dusti Sloan Tulsa Community College, Department of Biotechnology Abstract More than one half of the people living in the United States take dietary supplements. However, the Dietary Supplement Health and Education Act of 1994 mandated that dietary supplements were to be regulated as food rather than drugs, leading to less testing overall of dietary supplements. Hence, the present study aims to determine the toxicity of two dietary supplements, Cellucor C4 and Astaxanthin. Toxicity was determined by performing an MTT viability assay on CHO cells. An ANOVA showed that there was a statistically significant decrease in the viability of the cells grown in media containing 1.25% and 2.5% Astaxanthin. The results for the C4 compound were inconclusive. Overall, Astaxanthin has very little toxic effect on CHO cells and the MTT assay is not effective using the Cellulcor C4 dietary supplement. Introduction The Food and Drug Administration (FDA) is responsible for ensuring that dietary supplements are not misbranded or adulterated in any form. Supplements are often marketed to have medicinal properties even though manufacturers are not allowed to make false claims that their product can cure or treat disease. Federal laws, however, do not require that these supplements be proven safe by FDA standards before being marketed to the public. Pharmaceuticals, on the other hand, are required to undergo rigorous safety testing prior to being released for public use. Cellucor C4 is a pre- workout supplement that contains a mixture of vitamins, caffeine, theacrine, creatine nitrate, B-alanine, L-tyrosine, arginine, and other proprietary ingredients. Astaxanthin, a carotenoid pigment, purportedly acts as an antioxidant to help boost the immune system, prevent heart attack, cancer, Alzheimer’s and Parkinson’s. We used Chinese Hamster Ovary (CHO) cells to determine whether these dietary supplements affect cell viability using an MTT assay. Methods Results Acknowledgement We would like to extend our gratitude to Tulsa Community College for their strong support, in particular, the Southeast Campus Science and Mathematics Division. Thank to Oklahoma INBRE for their continued support and for funding this project. We determined the viability of CHO cells after exposure to two dietary supplements, Cellucor C4 and Astaxanthin. The MTT result using Cellucor C4 was inconclusive because the dietary supplement itself altered the color of the MTT Assay. Another assay would need to be conducted to further assess whether Cellucor C4 is toxic to cells. However, the images on day 1 indicate that the cells were viable in all Cellucor C4 concentrations. The cells grown in Astaxanthin concentrations less than 1.25% were not different from control. Cells grown in 2.5% or 1.25% Astaxanthin were significantly less viable than control cells suggesting that the References Future Work Conclusion Built in controls were incorporated and served to eliminate confounding, extraneous variables. • Each assay plate contained negative controls that consisted of media only, with or without the supplements. • In addition, positive controls served to verify that our cell stock was healthy. After the 5 day incubation period, MTT dye was added, and absorbances were obtained using a microplate spectrophotometer set at 595nm. Viable cells turn the yellow tetrazoleum salt of the MTT assay to a purple formazan. Results were interpreted using • Tube 1: 65mls 10% Cellucor dilution Add 30ml to tube #2. Mix until homogenous. • Tube 2: 30ml of CGM • 5% Cellucor dilution Add 30 ml to tube #3. Mix until homogenous. • Tube 3: 30ml of CGM • 2.5% Cellucor dilution Add 30 ml to tube #4. Mix until homogenous. • Tube 4: 30ml of CGM • 1.25% Cellucor dilution Add 30ml to tube #5. Mix until homogenous. • Tube 5: 30ml of CGM • 0.625% Cellucor dilution Cellucor • Tube 1: 65 mls of 2.5% Astaxanth in stock Add 30ml to tube #2. Mix until homogenous. • Tube 2: 30ml of CGM • 1.25% Astaxanth in dilution Add 30 ml to tube #3. Mix until homogenous. • Tube 3: 30ml of CGM • 0.625% Astaxanth in dilution Add 30 ml to tube #4. Mix until homogenous. • Tube 4: 30ml of CGM • 0.3125% Astaxanth in dilution Add 30ml to tube #5. Mix until homogenous. • Tube 5: 30ml of CGM • 0.156% Astaxanth in dilution Astaxant hin Cellucor C4 MTT Assay 1 2 3 4 5 6 7 8 9 10 11 12 Cellucor C4 MTT Assay Graph 1. The viability of cells grown in Astaxanthin concentrations of 0.625%, 0.3125%, and 0.156% are not significantly different from control. The viability of cells grown in Astaxanthin concentrations of 2.5% and 1.25% demonstrate statistically low viability compared to control. Different capital letters indicate statistically significant differences. Figure 2. 96 well plate containing Cellucor C4. Columns 1-6: Media without cells in the following concentrations: Control, 10%, 5%, 2.5%, 1.25% and 0.625%. Columns 7- 12: Media with cells in the following concentrations: Control, 10%, 5%, 2.5%, 1.25% and 0.625%. The Cellucor C4 contains a substance that reacts with the MTT solution altering the color to purple in wells with and without cells. Assessing cell viability using the MTT assay did not yield accurate results. "FDA Dietary Supplements." FDA U.S. Food and Drug Administration. U.S Department of Health and Human Services, n.d. Web. 26 Oct. 2015. For each supplement, cells were plated in ten, 96-well plates at 5 x 10 4 cells/mL and incubated for 2 days in regular growth media. A 2-fold dilution series was employed from initial stock concentrations (Cellucor C4-10%, Astaxanthin-2.5%). Regular growth media was removed and the different concentrations of Cellucor C4 or Astaxanthin were added to the appropriate wells and incubated 5 days. Figure 1. 96 well plate containing Astaxanthin. Columns 1-6: Media without cells in the following concentrations: Control, 2.5%, 0.625%, 0.3125%, 0.156%. Columns 7- 12: Media with cells in the following concentrations: Control, 2.5%, 0.625%, 0.3125%, 0.156%. Figure 3. CHOK-1 cell growth in varying concentrations of Cellucor C4. Images were taken on day 1. Astaxanthin MTT Assay 1 2 3 4 5 6 7 8 9 10 11 12
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Transcript of Astaxanthin and Cellucor Poster Fall 2015
Cellucor C4• Learn what ingredients in
Cellucor caused the MTT assay to react in blank wells.
• Try another type of assay to avoid the MTT reaction.
• Test using different cell lines.
Astaxanthin• Vary the range of dilution. • Use solvent such as DMSO to allow
use of the whole supplement.• Find alternate forms of sterilization.
What CHO Cells Say About the Supplements, Cellucor C4 and AstaxanthinBrian Eccleston, Bipina Acharya, Ciara Boatright, Jordan Cox, Brandon Curry, Kameron Killam, Blake King,
Samantha Rice, Jennifer Yang, Dusti SloanTulsa Community College, Department of Biotechnology
Abstract
More than one half of the people living in the United States take dietary supplements. However, the Dietary Supplement Health and Education Act of 1994 mandated that dietary supplements were to be regulated as food rather than drugs, leading to less testing overall of dietary supplements. Hence, the present study aims to determine the toxicity of two dietary supplements, Cellucor C4 and Astaxanthin. Toxicity was determined by performing an MTT viability assay on CHO cells. An ANOVA showed that there was a statistically significant decrease in the viability of the cells grown in media containing 1.25% and 2.5% Astaxanthin. The results for the C4 compound were inconclusive. Overall, Astaxanthin has very little toxic effect on CHO cells and the MTT assay is not effective using the Cellulcor C4 dietary supplement.
Introduction
The Food and Drug Administration (FDA) is responsible for ensuring that dietary supplements are not misbranded or adulterated in any form. Supplements are often marketed to have medicinal properties even though manufacturers are not allowed to make false claims that their product can cure or treat disease. Federal laws, however, do not require that these supplements be proven safe by FDA standards before being marketed to the public. Pharmaceuticals, on the other hand, are required to undergo rigorous safety testing prior to being released for public use. Cellucor C4 is a pre-workout supplement that contains a mixture of vitamins, caffeine, theacrine, creatine nitrate, B-alanine, L-tyrosine, arginine, and other proprietary ingredients. Astaxanthin, a carotenoid pigment, purportedly acts as an antioxidant to help boost the immune system, prevent heart attack, cancer, Alzheimer’s and Parkinson’s. We used Chinese Hamster Ovary (CHO) cells to determine whether these dietary supplements affect cell viability using an MTT assay.
Methods Results
AcknowledgementWe would like to extend our gratitude to Tulsa Community College for their strong support, in particular, the Southeast Campus Science and Mathematics Division. Thank to Oklahoma INBRE for their continued support and for funding this project.
We determined the viability of CHO cells after exposure to two dietary supplements, Cellucor C4 and Astaxanthin. The MTT result using Cellucor C4 was
inconclusive because the dietary supplement itself altered the color of the MTT Assay. Another assay would need to be conducted to further assess whether Cellucor C4 is toxic to cells. However, the images on day 1 indicate that the cells were viable in all Cellucor C4 concentrations.
The cells grown in Astaxanthin concentrations less than 1.25% were not different from control. Cells grown in 2.5% or 1.25% Astaxanthin were significantly less viable than control cells suggesting that the supplement impacts the cell viability at those concentrations.
References
Future Work
Conclusion
Built in controls were incorporated and served to eliminate confounding, extraneous variables. • Each assay plate contained negative controls that
consisted of media only, with or without the supplements.
• In addition, positive controls served to verify that our cell stock was healthy.
After the 5 day incubation period, MTT dye was added, and absorbances were obtained using a microplate spectrophotometer set at 595nm. Viable cells turn the yellow tetrazoleum salt of the MTT assay to a purple formazan.
Results were interpreted using corrected absorbency [(media+cells) - blanks] and those values were then compiled and subsequently analyzed using ANOVA and the Neuman-Keuls post hoc statistical analyses.
• Tube 1: 65mls 10% Cellucor dilution
Add 30ml to tube #2. Mix until
homogenous.
• Tube 2: 30ml of CGM
• 5% Cellucor dilution
Add 30 ml to tube #3.Mix until
homogenous. • Tube 3: 30ml of CGM
• 2.5% Cellucor dilutionAdd 30 ml to tube #4.
Mix until homogenous.
• Tube 4: 30ml of CGM
• 1.25% Cellucor dilution
Add 30ml to tube #5.Mix until
homogenous.• Tube 5:
30ml of CGM
• 0.625% Cellucor dilution
Cellucor
• Tube 1: 65 mls of 2.5% Astaxanthin stock
Add 30ml to tube #2. Mix until
homogenous.
• Tube 2: 30ml of CGM
• 1.25% Astaxanthin dilution
Add 30 ml to tube #3.Mix until
homogenous. • Tube 3: 30ml of CGM
• 0.625% Astaxanthin dilutionAdd 30 ml to tube #4.
Mix until homogenous.
• Tube 4: 30ml of CGM
• 0.3125% Astaxanthin dilution
Add 30ml to tube #5.Mix until
homogenous.• Tube 5:
30ml of CGM
• 0.156% Astaxanthin dilution
Astaxanthin
Cellucor C4 MTT Assay
1 2 3 4 5 6 7 8 9 10 11 12
Cellucor C4 MTT Assay
Graph 1. The viability of cells grown in Astaxanthin concentrations of 0.625%, 0.3125%, and 0.156% are not significantly different from control. The viability of cells grown in Astaxanthin concentrations of 2.5% and 1.25% demonstrate statistically low viability compared to control. Different capital letters indicate statistically significant differences.
Figure 2. 96 well plate containing Cellucor C4. Columns 1-6: Media without cells in the following concentrations: Control, 10%, 5%, 2.5%, 1.25% and 0.625%. Columns 7-12: Media with cells in the following concentrations: Control, 10%, 5%, 2.5%, 1.25% and 0.625%. The Cellucor C4 contains a substance that reacts with the MTT solution altering the color to purple in wells with and without cells. Assessing cell viability using the MTT assay did not yield accurate results.
"FDA Dietary Supplements." FDA U.S. Food and Drug Administration. U.S Department of Health and Human Services, n.d. Web. 26 Oct. 2015.
For each supplement, cells were plated in ten, 96-well plates at 5 x 104 cells/mL and incubated for 2 days in regular growth media.
A 2-fold dilution series was employed from initial stock concentrations (Cellucor C4-10%, Astaxanthin-2.5%).
Regular growth media was removed and the different concentrations of Cellucor C4 or Astaxanthin were added to the appropriate wells and incubated 5 days.
Figure 1. 96 well plate containing Astaxanthin. Columns 1-6: Media without cells in the following concentrations: Control, 2.5%, 0.625%, 0.3125%, 0.156%. Columns 7-12: Media with cells in the following concentrations: Control, 2.5%, 0.625%, 0.3125%, 0.156%.
Figure 3. CHOK-1 cell growth in varying concentrations of Cellucor C4. Images were taken on day 1.
Astaxanthin MTT Assay1 2 3 4 5 6 7 8 9 10 11 12
