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 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
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%
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
dilution
Add 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 Assay
1 2 3 4 5 6 7 8 9 10 11 12