James Constantin Central Catholic High School Grade 11

 Basic Definition- cell that can produce lineages more specialized than themselves and can renew itself  Spectrum of Stem Cell Behavior:  totipotent embryonic cells  pluripotent cells  multipotent cells  unipotent cells.

 Subclone of the mus musculus (mouse) myoblast cell line.  Differentiates rapidly, forming contractile myotubes and produces characteristic muscle proteins.  Mouse stem cell line is used as a model in many tissue engineering experiments.  Useful model to study the differentiation of non-muscle cells (stem cells) to skeletal muscle cells.  Expresses muscle proteins and the androgen receptor (AR).  AR- DNA binding transcription factor which regulates gene expression.

 Whey protein is a mixture of globular proteins isolated from whey, the liquid material created as a by-product of cheese production. Some preclinical studies in rodents have suggested that whey protein may possess anti- inflammatory or anti-cancer properties; however, human data is lacking. The effects of whey protein on human health are of great interest and are currently being investigated as a way of reducing disease risk (such as muscle degeneration), as well as a possible supplementary treatment for several diseases. Whey protein is commonly marketed and ingested as a dietary supplement, and various health claims have been attributed to it in the alternative medicine community.

 Whey is essential in the bodybuilding world today because of its ability to be digested very rapidly. This allows the protein to become available for muscle building very quickly. Most commonly it is used after workouts to help increase levels of amino acids in the blood, which are taken up by the muscles to ultimately increase mass. In addition, during exercise, whey helps open up blood flow by inhibiting an angiotensin-converting enzyme which originally constricts blood vessels; this allows better flow of nutrients to necessary areas to help repair and rebuild muscle tissues. The use of whey protein as a source of amino acids and its effect on reducing the risks of diseases such as heart disease and cancer is the focus of ongoing research.

 To examine the effect(s) of (GNC) Wheybolic Extreme on proliferation and differentiation of C2C12 murine adult stem cells

Null: GNC Wheybolic Extreme will not have an effect on the proliferation, differentiation, and survivorship of C2C12 cells. Alternate: GNC Wheybolic Extreme will have an effect on the proliferation, differentiation, and survivorship of C2C12 cells. Specifically the variable will promote growth and promote the cells to differentiate into myofibrils.

 Cryotank  Three 75mm 2 tissue culture treated flasks  Twenty-four 25 mm 2 tissue culture treated flasks  10% fetal bovine serum  C2C12 Myoblastic Stem Cell Line  Trypsin-EDTA  Pen/strep  Macropipette + sterile macropipette Tips (1 mL, 5 mL, 10, mL, 20 mL)  Micropipettes + sterile tips  DMEM media -1% and Complete Media (4 mM L- glutamine, 4500 mg/L glucose, 1 mM sodium pyruvate, and 1500 mg/L sodium bicarbonate + [ 10% fetal bovine serum for complete])  75 mL culture flask  Incubator  Aspirating Vacuum Line  Nikon Inverted Compound Optical Scope  Laminar Flow Hood  Laminar Flow Hood UV Sterilizing Lamp  Labeling Tape  GNC Amplified Wheybolic Extreme 60  Hemocytometer  Sterile PBS  Ethanol (70% and 100%)  Distilled water  Antibody staining system  Fluorescent sensitive microscope Hemocytometer grid

 A 1 mL aliquot of C2C12 cells from a Cryotank was used to inoculate 30 mL of 10% serum DMEM media in a 75mm 2 culture flask yielding a cell density of approximately 10 6 to 2x10 6 cells.  The media was replaced with 15 mL of fresh media to remove cryo-freezing fluid and incubated (37° C, 5% CO 2 ) for 2 days until a cell density of approximately 4x10 6 to 5x10 6 cells/mL was reached.  The culture was passed into 3 flasks in preparation for experiment and incubated for 2 days at 37° C, 5% CO 2.

Day 1  After trypsinization, cells from all of the flasks were pulled into 1 common 75mm 2 flask (cell density of approximately 1 million cells/mL).  5 ml of the cell suspension was added to 6 25 mm 2 ( 2 for each grouping) tissue culture treated flasks, creating a cell density of approximately 10 5 cells per flask.  The following concentrations of variable (next page) were added to the flasks. 4 flasks for each group (2 for proliferation, 2 for differentiation)  Using one flask from each group, cell densities were determined as follows: ▪ The cells were trypsinized and collected into cell suspension. ▪ 25 µl aliquots were transferred to a Hemacytometer for quantification (six counts per flask). Day 2 and Day 5  Using the Nikon Inverted Microscope, images of several representative areas of each flask were taken.

 3mL of the cell suspension was added to three glass bottom well plates  The plates were incubated for one day  The following day (Day 1) 30 µL of variable was added to the two variable plates  The plates were allowed to incubate for one week until myotubules formed 1. The three plates were fixed by washing with PBS and fixing with ice-cold methyl alcohol (MeOH) 2. Stain plates with primary and secondary GFP, Desmin, and Dapi 3. Images were recorded utilizing a fluorescent inverted view microscope

Mini Flasks (Proliferation) 2 flasks per group Plates (Differentiation) 3 plates per group 0.0%0 ml’s of 1%stock (Wheybolic Extreme) 5 ml’s Media 0 ml’s of 1% stock (Wheybolic Extreme) 3ml’s 0.01%50 microliters of 1% stock (Wheybolic Extreme) 4.95 ml’s Media 3 microliters 1% stock (Wheybolic Extreme) 2.95 ml’s Media 0.001%5 microliters of 1% stock (Wheybolic Extreme) ml’s Media 3 microliters 1% stock (Wheybolic Extreme) ml’s Media

 ANOVA: In statistics, analysis of variance is a collection of statistical models, and their associated procedures, in which the observed variance in a particular variable is partitioned into components attributable to different sources of variation. In its simplest form ANOVA provides a statistical test of whether or not the means of several groups are all equal, and therefore generalizes t-test to more than two groups  Differentiantiation a Myosin Quantification Template was used: Overlaps images from fluorescent scope and compares nuclei, myotube growth etc.By comparing the means you are comparing variation between the groups to the variation within the groups.  Dunnett's test compares each variable group individually to the control group to see if that particular variable caused them to vary significantly. A T-test will yield the same p-value as the ANOVA and therefore will show insignificant results.

Control Day 3 Day % 0.01% Proliferation

Variable Concentration% DifferentiationNuclei/Myotube Control22.645% % %2.572 An ANOVA was performed on the data, and the f-value was larger than the f-crit, so the results are SIGNIFICANT.

Normal Dapi Stain Desmin Stain GFP Stain

 Null hypothesis can be accepted for the parameters that GNC Wheybolic Extreme does not have an effect on C2C12 stem cell survivorship.  Null hypothesis can be accepted for the parameters that GNC Wheybolic Extreme does not have an effect on C2C12 stem cell proliferation on Day 1.  The null Hypothesis can be accepted for the parameters that GNC Wheybolic Extreme does not have an effect on C2C12 stem cell proliferation on Day 2.  Differentiation Experiment  The Null was rejected for both variable concentrations. Supporting previous studies that Whey protein promotes muscle growth Between DaysDay 1 ProliferationDay 2 Proliferation

 Different cell lines 3T3  Possibility of Errant cell count  Over trypsinization  Damages cells and has a negative effect on colony health  Trypan Blue Dye Exclusion Assay ▪ Tells if cells are alive so gives more quantifiable information  CyQUANT™ Cell Proliferation Assay ▪ More quantitative than counting cells on a Hemocytometer ▪ Fluorescent dye binds to nucleic acid in the cell

 John Wilson, Biostatistician for the University of Pittsburgh  Conrad M. Zapanta, Ph.D BiomedicalEngineering Laboratory, Carnegie Mellon University  Mark Krotec, PTEI  ive_stress/oxidativestress.htm     Dr. Duprimia CMU