LAB MANUAL 1: MUSCLE DIFFERENTIATION

NOM: Anna Aguilera Romero

NIA: 241412
GRUP DE PRÀCTIQUES: 101

LAB MANUAL 1: MUSCLE DIFFERENTIATION

PR Content Class Duration (h)
1.1 - Presentation & Introduction. Lab 2,5h
- Trypsinization and Counting. 61.211-213
- Subculture of the three six-well C2C12 plates, one for the proliferation
assay and two for the differentiation and fusion assays.
1.2 - Microscopic observation, Counting, changing GM medium and adding Lab 1h
TSA. 61.211-213
1.3 - Microscopic observation, Counting. Lab 1h
- Changing GM and DM mediums. Adding LY294002. 61.211-213
1.4 - Microscopic observation Lab 2h
- Proliferation assay: Counting (Calculate the cell number per day 61.211-213
and organize the values. A graph will be done during the 1.8 class).
Also recollect cell lysates from the proliferation plate.
- Differentiation assay: Change medium (fresh DM and add
again LY294002)
1.5 - Microscopic observation. Lab 2,5h
- Selection of plates for each assay. 61.211-213
- In one of the differentiation assay plates start the
immunocytochemistry against MHC (blocking & primary antibody).
- In the other differentiation plate recollect cell lysates for the MCK assay -
- Quantification of protein (Bradford) of cell lysates and freeze them.
1.6 - Finish immunocytochemistry against MHC (secondary antibody & Lab 2,5h
develop). 61.211-213
- Results of the immunocytochemistry (photos of representative fields
will be taken by the Cell Biology professors at the PRBB) will be used to
determine differentiation and fusion indexes during the 1.8 class.
1.7 - Measurement of MCK activity in frozen cell lysates.

1.8 - Analysis and interpretation of the data obtained in the lab. Computer 3h
room
Informative note:
As you can see in the above table the “Practice 1: Muscle Differentiation” consists in 8 sessions. In
order to understand all the practice procedure, you will have to read and answer all the questions
of the laboratory manual. The results of this lab work will be used for the “Practical Research
Project” (PRP) activity. This laboratory manual will be collected at the end of the course with all the
calculations/answers/comments. Graphs performed at the data analysis session may be attached.

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INTRODUCTION
Cellular differentiation is the process by which a cell becomes increasingly specialized in form and
function. The classic example is the process by which a zygote develops from a single cell into a
multicellular embryo that further develops into a more complex fetus. Cellular differentiation is
regulated by cell contact and extracellular factors. We have selected the muscle tissue as a
paradigm of the differentiation process. One of the best cellular models for muscle differentiation
are the C2 mouse myoblast cell line (Yaffe D and Saxel O, Nature 1977), and the C2C12 (produced
by H. Blau, et al., Science 1985) a subclone of the C2.
C2C12 cells when cultured in the presence of growth factors (i.e. culture medium with fetal bovine
serum –FBS-) proliferate and stay in a non-differentiated phenotype. However, when they are
cultured in medium devoid of growth factors (with low concentration of FBS or with other less
potent serums) or reach “confluence”, they exit the cell cycle and begin to differentiate (which is
accompanied by a significant increase of expression levels of MyoD and other muscle bHLH
proteins and their target genes). Soon after, the cells fuse to form multinucleated myotubes.
Therefore, C2C12 cells represent an excellent model to study muscle differentiation and to analyze
the mechanisms of proliferation, cell cycle exit and activation of the myogenic gene expression
program.

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Aims
a) Phenotypical and biochemical evaluation of the proliferation and differentiation of
muscle cells
b) Analysis of the signaling cascades involved in the differentiation of muscle cells
Methodology
Cell line: C2C12 (mouse myoblasts).
Methods:
 Microscopic determination of cell number and myotube formation
 Expression of differentiation-specific proteins: Immunocytochemistry analysis of Myosin Heavy
Chain (MHC) expression
 Expression of differentiation-specific proteins: Biochemical analysis of muscle Creatine Kinase
(MCK) activity. MCK is an enzyme that catalyzes the transfer of one phosphate group from ATP
to creatine generating phosphocreatine, an important energy reservoir in muscle. In this assay,
MCK activity is determined by a coupled enzyme reaction resulting in the production of
NADPH, measured at 340 nm, proportionate to the CK activity present in the sample.
Culture media:
1. Proliferation medium (Growth medium o GM): DMEM, Fetal bovine serum (FBS) 10 %, L-
glutamine, penicillin/streptomycin
2. Differentiation medium (DM): DMEM, Horse serum (HS) 2 %, L-glutamine,
penicillin/streptomycin and ITS (insulin-transferrin-selenium).
Solutions:
1.
PBS (Phosphate buffered saline): 0.133 M NaCl, 8.6 mM K 2HPO4 and 1.5 mM KH2PO4, pH
7.6.
2.
Lysis Buffer: 50 mM HCl-Tris pH 8, 150 mM NaCl, 0,1% Nonidet P-40, 5 mM EGTA, 5 mM
EDTA, 20 mM NaF. Protease inhibitors (SIGMA. Contains Aprotinin, Bestatin, E-64, Leupeptin
and Pepstatin A).
3.
Blocking solution (TNB). Buffer containing high protein concentration which allows to
clock unspecific binding of the antibodies: 0.1 M Tris-HCl, pH 7.5, 150 mM NaCl and 0.5%
Blocking Reagent (from TSA Kit - Perkin Elmer).
4.
ABC solution: A mixture of solution A (Avidin DH) and solution B (Biotinylated Horseradish
Peroxidase H) from the ABC kit (Vector Laboratories).
5.
Developing solution: A mixture of DAB (3,3'-Diaminobenzidine; SIGMA) 0.7 mg/ml and the
peroxidase substrate H2O2 1.6 mg/ml. Mix both components before use (60 l of DAB 50
mg/ml and 2 l of H2O2 30% in 10 ml PBS).
6.
MCK activity reagent (CK-NAC Reagent, Thermo): contains all the needed enzymes and
substrates for the coupled enzyme reaction. Dissolve in 10 ml water before use.
Antibodies:
1. MHC: MF-20 mouse neat hybridoma supernatant (Developmental Studies Hybridoma Bank).
2. Secondary: Biotinylated Goat anti–mouse (Jackson ImmunoResearch Laboratories)

Suppliers:
Bradford reagent: Bio-Rad; Trichostatin A: SIGMA; LY294002: CalBiochem; DMEM: Gibco

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Microscope details:

Digital images will be acquired in a DMR6000B (Leica) microscope equipped with a HCX PL Fluotar
20X/0.50 objective and a DFC300FX camera using the Leica Application software (Leica).

Waste Disposal:
Biological waste
Solid waste generated in the cell culture hood or bench (i.e. pipettes, tips, plates) should be disposed
in the Biological waste containers (Black container with Yellow Lid)

Liquid waste in the cell culture hood or bench (i.e. Culture medium, cell residues, PBS, PBS-T)
should be disposed in a glass inside the hood during the work, and afterwards should be disposed
in a tank which will be labeled accordingly.
The paper and plastic wrappings of the cell culture material (i.e. pipettes, plates) is not biological
waste and should be disposed with the normal waste containers in the lab.
Chemical waste
During the immunochemistry. Formaldehyde used for fixation should be disposed in a 50 ml
conical tube that will be given to each group, after the class the residues will be collected by the
professor.
Toxic/Cytotoxic waste
Cuvettes with Bradford reagent should be disposed in the small container in the bench, then will
be disposed in the Toxic waste container (Blue container with Black lid).
During the MCK activity assay.
DAB is a potent mutagenic agent. Liquids with DAB should be disposed in a 50 ml conical tube that
will be given to each group, after the class the residues will be collected by the professor.

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QUICK SCHEDULE
1.1:
1. Introduction.
2. Microscopic observation of the C2C12 cells.
3. Trypsinization and subculture of the cells in proliferation medium (GM), a rich medium with
high concentration of growth factors.
Each group will seed three six-well plates, one for the proliferation assay and two for the
differentiation and fusion assays.
Proliferation assay:
A 7000 cells B 7000 cells C 7000 cells
D 15.000 cells E 15.000 cells F 15.000 cells

Differentiation & Fusion assay (two plates):

A 100.000 cells B 100.000 cells C 100.000 cells
D 200.000 cells E 200.000 cells F 200.000 cells

1.2:
1. Microscopic observation of the C2C12 cells.
2. Cell number: Cells will be counted in the proliferation assay plate. Cells will be counted at
the microscope, extrapolating the number of cells present in several 20X fields to the well
surface.
3. Change medium, adding fresh GM medium. TSA (Trichostatin A) at a final concentration
of 50 nM will be added to two wells per plate (one of each cell density).

1.3:
1. Microscopic observation of the C2C12 cells.
2. Cell number: Cells will be counted in the proliferation assay plate. Cells will be counted at
the microscope, extrapolating the number of cells present in several 20X fields to the well
surface.
3. Change medium, adding fresh medium.
a. In the proliferation assay, use GM.
b. In the differentiation assay, we will change medium to differentiation medium (DM)
to allow cells to differentiate over the weekend.
4. Add LY294002 inhibitor at a final concentration of 5 M to two wells per plate (one of each
cell density).
Cell will differentiate and fuse forming myotubes.

1.4:
1. Microscopic observation of the C2C12 cells.
2. Cell number: Cells will be counted in the proliferation assay plate. Cells will be counted at
the microscope, extrapolating the number of cells present in several 20X fields to the well
surface.
3. Calculate the cell number per day and organize the values of the proliferation assay. A
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 graph will be done during the 1.8 class.

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 4. Cells will be lysed, and lysates will be collected from some of the proliferation assay wells to
be used as negative control for the MCK assay.
5. In the differentiation assay, change medium to fresh DM. Add again LY294002 inhibitor to
the correct wells.

1.5:
1. Microscopic observation of the C2C12 cells.
2. In one of the differentiation assay plates, cells will be lysed, and lysates will be collected for
the MCK assay.
3. Determination of the protein concentration of cell lysates collected for the MCK assay using
the Bradford methods.
a. Standard curve with BSA.
b. Quantification of the protein concentration of the cell lysate using the standard
curve as reference.
Keep lysates frozen at -20ºC until tomorrow
4. In the other differentiation assay plate, some wells will be selected to begin the
immunocytochemistry against MHC.

1.6 & 1.7:

1. Immunocytochemistry against MHC, second part.
2. MCK activity assay.

1.8- Computer room:

1. Results of the immunocytochemistry (photos of representative fields will be taken by the
Cell Biology professors at the PRBB) will be used to determine differentiation and fusion
indexes.
2. Analysis and interpretation of the data obtained in the lab (computer room).
3. Perform the proliferation assay graphs.

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 1.1-

1. Introduction.
Each lab group will be divided in 5 subgroups. Two 150 mm plates with C2C12 cells will be given to
each group.
2. Microscopic observation of the C2C12 cells.
Using the lab microscopes with the Phase Contrast configuration, observe the C2C12 cells and
familiarize with their proliferative phenotype (myoblasts). Write down your observations.
OBSERVATIONS

We observed little round cells with one single nuclei.

3. Trypsinization and subculture of the cells in proliferation medium (GM), a rich medium
with high concentration of growth factors. Remember that cells should have strict aseptic
conditions:
 Before beginning the lab work the laminar flow hood should be turn on and proper flow confirmed

 Before and after use, the hood surface should be disinfected thoroughly, and the surrounding
areas and equipment should be cleaned routinely. Use 70 % ethanol.

 Wash your hands before and after working with cell cultures. Use gloves. Always wipe your hands
and your work area with 70% ethanol.

 Thaw trypsin. Pre-heat the culture media at 37ºC in a water bath.

 Avoid pouring media and reagents directly from bottles or flasks.

 Use sterile glass or disposable plastic pipettes and a pipettor to work with liquids and use each
pipette only once to avoid cross contamination. Do not unwrap sterile pipettes until they are to be
used. Keep your pipettes at your work area.

 You will need two sterile glasses, one for solid residues (pipette tips) and one for liquid residues
(culture medium, PBS).

Trypsinization:
1. Remove the media from the plate.
2. Wash the plate containing cells with 10 ml PBS 1x during some seconds. Remove PBS. Note:
Trypsin is inhibited by components of the serum, so washing with PBS is mandatory. Remove
PBS after washing to avoid dilution of the trypsin

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3. Add 3 ml of Trypsin. Incubate for 5 minutes at 37ºC in the incubator. Check that cells are
floating at the microscope. If rounded but still not floating, tap the plate gently and make sure
the cells are floating.
4. Add 7 ml of GM to the plate. This will stop trypsin action and allow better transfer. Transfer the
whole volume to a 15 ml centrifuge tube.
5. Spin at 1000 rpm for 5 minutes.
6. Remove the supernatant by gently decanting the liquid out.
7. Resuspend the cell pellet in 10 ml of GM. Pipette gently up and down until full resuspension.
Mix well to separate clumps, but be gentle with the cells and avoid bubbles. This is our cell
solution to be used for counting and to calculate the volume needed to seed each cell density
per well.
8. Each group will seed three six-well plates, one for the proliferation assay and two for the
differentiation and fusion assays. Medium volume per well is 2 ml.
9. Proliferation assay:
A 7000 cells B 7000 cells C 7000 cells
D 15.000 cells E 15.000 cells F 15.000 cells

10. Differentiation & Fusion assay (two plates):

A 100.000 cells B 100.000 cells C 100.000 cells
D 200.000 cells E 200.000 cells F 200.000 cells

COUNTING CELLS IN SUSPENSION: THE HEMOCYTOMETER OR NEUBAUER CHAMBER

For microbiology, cell culture, and many applications that require use of suspensions of cells it is
necessary to determine cell concentration. A device used for determining the number of cells per
unit volume of a suspension is called a counting chamber. The most widely used type of chamber is
called a hemocytometer, since it was originally designed for performing blood cell. One entire grid
on standard hemocytometers with Neubauer rulings can be seen at 40x (4x objective).
The main divisions separate the grid into 9 large squares. Each square has a surface area of 1 mm 2,
and the depth of the chamber is 0.1mm. Each square of the hemocytometer (with a cover slip in
place) represents a total volume of 0.1 mm 3 or 10-4 cm3. Since 1 cm3 =1 ml, the calculated cell
concentration will be in cells per ml.
You may count the cells in the four corner squares (L). A common convention is to count cells that

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touch the middle lines (of the triple lines) to the left and top of the square, but do not count cells

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similarly on the right or bottom of the square (to avoid double-counting). For cells that overlap a
line, count a cell as "in" if it overlaps the top or right line, and "out" if it overlaps the bottom or left
line. To get the final count in cells/ml:
Cells per ml = the average count per square x the dilution factor x 104
On top of counting the cell number, we will determine the cell viability. One of the earliest and
most common methods for measuring cell viability is the trypan blue (TB) exclusion assay. Trypan
blue is a ~960 Daltons molecule that is cell membrane impermeable and therefore only enters cells
with compromised membranes. The trypan blue exclusion assay allows for a direct identification
and enumeration of live (unstained) and dead (blue) cells in a given population.
Cell counting:
1. In a 1.5 ml tube, prepare a 50µl 1:4 dilution of the cell suspension using trypan blue (30 µl
of trypan blue solution and 10 µl of the cell suspension).
2. Pipette up and down several times to obtain a homogenous suspension.
3. Put 10 µl of this suspension in the hemocytometer. The cell suspension is introduced into
one of the V-shaped areas. Allow the area under the coverslip to fill by capillary action.
Enough liquid should be introduced so that the surface under the cover slip is just covered.
The counting chamber is then placed on the microscope stage and the counting grid is
brought into focus at low power.
4. Count cells in the four corner squares (L) and calculate the number of cells per ml taking
into account the trypan blue dilution factor.
5. CALCULATIONS:

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 1.2-

1. Microscopic observation of the C2C12 cells. Using the lab microscopes with the Phase
Contrast configuration.
a. In the differentiation assay plates, observe the C2C12 cells and estimate the % of
confluence in each well.
b. In the proliferation assay plate, count the cell number per well. Cells will be counted at the
microscope, extrapolating the number of cells present in several 20X fields to the well
surface. The surface of a well is 9.6 cm2, the surface of a 20X field is 0.00785 cm2.
2. Change medium, adding fresh GM medium. Medium volume per well is 2 ml.

3. TSA (Trichostatin A) treatment. Add TSA at a final concentration of 50 nM to two wells per
plate (one of each cell density). Calculate the volume to add per well using a stock solution of
50 M. Medium volume per well is 2 ml.

CONFLUENCY

In cell culture biology, confluency is the term commonly used as a measure of the number of the cells in a cell cu

QUESTIONS:
-What is TSA and what is it used for?

TSA is a histone deacetylase (HDAC) inhibitor that chelates zinc ions present in the active site of
HDAC. It prevents of unpacking the DNA and inhibits its transcription affecting the gene
expression. TSA has anti-proliferative activity but promote differentiation.

-If you want a final concentration of 50 nM TSA, calculate the volume of TSA to add per well (2ml) using

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-Quote three examples of “relevant" publications where TSA has been used to study muscle differentia

https://pubmed.ncbi.nlm.nih.gov/22019851/
https://pubmed.ncbi.nlm.nih.gov/31171081/
https://pubmed.ncbi.nlm.nih.gov/23284002/

1.3-

1. Microscopic observation of the C2C12 cells. Using the lab microscopes with the Phase
Contrast configuration.
a. In the differentiation assay plates, observe the C2C12 cells and estimate the % of
confluence in each well.
b. In the proliferation assay plate, count the cell number per well. Cells will be counted at
the microscope, extrapolating the number of cells present in several 20X fields to the
well surface. The surface of a well is 9.6 cm2, the surface of a 20X field is 0.00785 cm2.
2. Change medium.
a. In the proliferation assay, add fresh GM medium.
b. In the differentiation assay, we will change medium to differentiation medium (DM) to
allow cells to differentiate over the weekend. Remember to wash with PBS to avoid
carrying on serum from the GM medium.
3. Treatment with LY294002 inhibitor: Add LY294002 inhibitor at a final concentration of 5 M
to two wells per plate (one of each cell density). Calculate the volume to add per well using a
stock solution of 5 mM. Medium volume per well is 2 ml.
We will have then in each plate: two Wells with no treatment (Control), two wells that had a
transient 24h treatment with TSA and two wells with treatment LY294002.

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QUESTIONS:

-What is LY294002 and what is it used for?

LY294002 is a strong inhibitor of PI3K. This affects the proliferation and the differentiation of
muscle cells.

-If you want a final concentration of 5M LY294002, calculate the volume of LY294002 to add
per well (2ml) using a stock solution of 5mM.

-Quote one or more examples of “relevant" publications where LY294002 has been used to
study muscle differentiation:

https://www.sciencedirect.com/science/article/pii/S0021925819843429
https://pubmed.ncbi.nlm.nih.gov/8702591/
https://pubmed.ncbi.nlm.nih.gov/17688959/

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 1.4-

1. Microscopic observation of the C2C12 cells. Using the lab microscopes with the Phase
Contrast configuration.
a. In the differentiation assay plates, observe the C2C12 cells and estimate the % of
confluence in each well.
b. In the proliferation assay plate, count the cell number per well. Cells will be counted at
the microscope, extrapolating the number of cells present in several 20X fields to the
well surface. The surface of a well is 9.6 cm2, the surface of a 20X field is 0.00785 cm2.

2. Preparation of cell lysates for the MCK assay. We will recollect the cells of two wells of the
Proliferation assay to use them as negative controls in the MCK activity assay. All this procedure
will be done on ice:
a. Take out the medium and wash twice with PBS.
b. Over the ice, take out PBS and add 300 µl of lysis buffer. Keep on ice 3 minutes,
occasionally swirling the plate to keep solution evenly spread. Use cell scraper to
scrape off cells. Pass the cell lysate through pipette several times to form a
homogeneous lysate. Transfer lysate to a cold 1.5 ml microcentrifuge tube on ice.
c. Centrifuge the lysate at 13.000 rpm for 3 minutes to separate the total protein
(supernatant) from the cellular debris (pellet).
d. Transfer supernatant to a new cold tube for further analysis. Label them as GM.
e. Freeze at -20 ºC.
3. Change medium in the differentiation assay. Add fresh DM. Maintain the LY294002
treatment in those two well.

1.5-

1. Microscopic observation of the C2C12 cells. Using the lab microscopes with the Phase
Contrast configuration check for myotube formation and choose the plate with bigger
myotubes to do the immunocytochemistry. The other plate will be used for the MCK activity
assay.

2. Preparation of cell lysates for the MCK assay. Collect the cell lysates from the selected plate
and do the same procedure as section 1.4.

3. Determination of the protein concentration of the cell lysates: We will use the Bradford
method. First, we will do a standard curve with BSA.
a. Preparation of protein standards with BSA. We will use a 1 mg/ml stock solution of
BSA to generate a standard curve. The linear range of these assays for BSA is 1.25–
10 µg/ml.

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 Indicated µl of BSA standard + 1 ml Bradford reagent
Mix
Measure the absorbance of the standards at 595nm.
BSA stock
BSA (g/ml) ABS (595nm)
(µl)
0,0 0 0
1 1 0,078
2 2 0,169
3 3 0,208
4 4 0,296
5 5 0,369
8 8 0,575
10 10 0,648

a. Standard curve: With the average absorbance values and the concentration of protein we
will do a standard curve where we may interpolate the concentration of protein in our
samples.
Standard curve

b. Preparation of the samples: Maintain the samples on ice.

2 or 10 µl of sample solution + 1 ml of Bradford reagent
Measure the absorbance of the samples at 595nm.
Protein concentration
Sample volume ABS (595nm)
(mg/ml)
GM 2 µl 0,265 0,018338
GM2 2 µl 0,338 0,023389
DM 2 µl 0,173 0,0119716
DM2 2 µl 0,367 0,0253964
TSA 2 µl 0,021 0,0014532
TSA2 2 µl 0,086 0,0059512
LY 2 µl 0,095 0,006574
LY2 2 µl 0,274 0,0189608

In case of being out of the standard curve, prepare higher or lower amount of the samples.

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4. Immunocytochemistry against MHC (myosin heavy chain), first part.
One of our aims is to evaluate muscle differentiation properly. Complementary to the MCK activity
assay, we will quantify differentiation and fusion by the detection of a myotube-specific protein by
immunocytochemistry, the MHC.
To locate the MHC antigen, the antigen must first be "fixed" in the cells. This is accomplished using
a fixative which crosslinks the MHC antigen molecules, rendering them insoluble and holding them
in their "natural" place.
The antigen-antibody complex formed is then identified using a previously labeled secondary
antibody that recognizes the specific antibody. In our case, we will use a biotinylated secondary
antibody with allow detection using an amplification system with the avidin-biotin system (AB kit).
In the ABC system, avidin is bound to a biotinylated peroxidase enzyme (HRP). Addition of H 2O2
combined with the chromogenic molecule Diaminobenzidine, DAB) produce an insoluble brown
product and H2O in the area where the antibody has bound the antigen.
Before performing the immunocytochemistry, the cells must be treated with a fixative solution that
immobilizes, stop autolytic processes and preserve the cells. This process is based on the
establishment of covalent bonds between molecules, such that they are stabilized and locked in
position and original structure.

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Protocol:

This may be done at the bench.

1. Remove cell culture medium and wash with PBS
2. Fixation: Incubate the cells in 1 ml of formaldehyde 3.7 % at room temperature for 10 min.
3. Wash three times with PBS 1 min each wash.
4. Blocking: Incubate with 500 l of blocking solution (TNB) at room temperature for 1 h.
5. Primary antibody: Incubate with 500 l of diluted anti-MHC antibody in TNB (1:2000) at 4 ºC
overnight.

1.6-

Immunocytochemistry against MHC (myosin heavy chain), second part.

6. Wash three times with PBS-Tween 0.1% (PBS-T). 1 min each wash.
7. Secondary antibody: Incubate the cells with 500 l of diluted biotinylated anti-mouse
antibody in PBS-Tween 0,1% (1:250) at room temperature for 45 minutes.
8. At the middle of the incubation, prepare the Vectastain ABC solution (10 l sol A + 10 l sol B +
480 l PBS per well). Prepare the full volume needed for all the wells and leave the solution
resting during 30 minutes at room temperature.
9. Wash three times with PBS-Tween 0.1% (PBS-T). 1 min each wash.

10. ABC: Incubate the cells with the Vectastain ABC solution at room temperature for 20 min.
11. Wash three times with PBS-Tween 0.1% (PBS-T). 1 min each wash. Wash two times with PBS.
Quick washes.
12. Develop: Add 500 l of Developing solution (60 l of DAB 50 mg/ml and 2 l of H2O2 30% in 10
ml PBS). Monitor reaction progress under the microscope. Reaction may proceed for 5 minutes.
Stop reaction when myotubes’ cytoplasm is staining in Brown but nuclei inside are still
unstained.
13. Stop reaction with water. Note: DAB-containing solutions are highly mutagenic and should
be disposed to a specific recipient, do not mix with other solutions.
14. Keep plates at 4ºC. The professor will take a full set of pictures of the data at the PRBB for
further analysis on 1.8 class.

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Conclusions:
From the observed results in the immunocytochemistry, summarize your conclusions:

1.7-

1. MCK activity assay.

One objective of the practice is to determine the extent of C2C12 cells differentiation in the
different experimental conditions. To do so, we will do a biochemical analysis of muscle Creatine
Kinase (MCK) activity, an enzyme present in myotubes but not in myoblasts. MCK is an enzyme that
catalyzes the conversion of creatine to phosphocreatine (an important energy reservoir in muscle)
using ATP and generating ADP. This reaction is reversible and thus ATP can be generated from
phosphocreatine and ADP.

In this assay, MCK activity is determined using a coupled enzymatic reaction resulting in the
production of NADPH. Amount of NADPH measured at 340 nm is proportional to the CK activity
present in the sample. In this reaction, phosphocreatine and ADP are converted to creatine and
ATP. The generated ATP is used by hexokinase to phosphorylate glucose resulting in glucose-6-
phosphate, which is oxidized by NADP in the presence of glucose-6-phosphate dehydrogenase to
produce NADPH and 6-phospho-D-gluconate. One unit of CK is the amount of enzyme that will
transfer 1.0 μmol of phosphate from phosphocreatine to ADP per minute at pH 6.0.

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We will use the kit CK-NAC (Thermo) which contains all the enzymes and substrates necessary for
the complete coupled reaction:

Each assay is performed with 800 µl of CK-NAC solution plus the sample. To allow the coupled
reaction to begin, each assay will be incubated 2 minutes at 37 ºC. The assays will be done one by
one, to avoid saturation of the reaction.
1. Blank. As blank we will use 40 µl of lysis buffer. This value will be our cero.
2. Afterwards we will prepare each sample (using 40 l per sample) one by one and annotate the
increment in absorbance every 30 seconds for 3 minutes. This will allow us to calculate the
increment in absorbance.

Since we do not know the precise amount of MCK enzyme present in each sample, the assay with
40 ml may have a lot of activity and arrive to saturation during the initial 2 minutes of incubation or
on the contrary stay below the minimal amount to be detected. In those cases, the assay should be
repeated with less or more amount of sample.

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 0 30’’ 60’’ 90’’ 120’’ 150’’ 180’’ Af-Ai U/ml
GM 0,03 0,047 0,058 0,067 0,079 0,089 0,1 0,07 0,078778
GM2 0,054 0,082 0,099 0,114 0,133 0,152 0,166 0,112 0,126045
DM 0,142 0,188 0,225 0,263 0,3 0,339 0,374 0,232 0,261093
DM2 0,461 0,636 0,728 0,829 0,921 1,016 1,109 0,648 0,729260
TSA 0,206 0,287 0,353 0,413 0,485 0,539 0,595 0,389 0,437781
TSA2 0,14 0,172 0,293 0,232 0,263 0,291 0,324 0,184 0,207073
LY 0,036 0,046 0,054 0,068 0,076 0,084 0,048 0,012 0,013504
LY2 0,011 0,019 0,021 0,025 0,03 0,035 0,04 0,029 0,032636

MCK activity calculation

𝑈 [(𝐴𝑓 − 𝐴𝑖)/3] × 𝑉𝑡
𝑀𝐶𝐾 ( )
=
𝑚𝑙 6.22 × 𝑉𝑠
We define each MCK unit (U) as the amount of enzyme able to produce 1 mmol of NADPH per
minute:
Af Absorbance final
Ai Absorbance initial
Vt Total Volume (in ml)
Vs Sample Volume (in ml)
6.22 Millimolar extinction coefficient of NADPH at 340 nm

3. To calculate the MCK specific activity (U/mg) we will divide the calculated MCK activity (U/ml)
by the concentration of protein.

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 Protein MCK activity
MCK specific
concentration U/ml
activity (U/mg)
(mg/ml)
GM 0,018338 0,07877813505 4,295895684
GM2 0,023389 0,1260450161 5,389072473
DM 0,0119716 0,2610932476 21,80938618
DM2 0,0253964 0,7292604502 28,7151112
TSA 0,0014532 0,4377813505 301,2533378
TSA2 0,0059512 0,207073955 34,79532783
LY 0,006574 0,01350482315 2,054277936
LY2 0,0189608 0,03263665595 1,721269986

4. Conclusions:
From the observed results in the MCK assay, summarize your conclusions.

BC Lab Manual 1 -2022-2023 22

 1.8-

Analysis and interpretation of the data obtained in the lab (computer room).

1.1- Proliferation assay

Calculate the cell number per day and organize the values with the data (1.2 & 1.3 & 1.4 sessions). A
graph will be done. Example:

1.2- Differentiation assay

1.2.1- MCK activity
Represent in a graph the MCK activity (U/ml) and MCK specific activity (U/mg). Example:

25
20
MCK (U/g)

15
10
5
0

A B C D

1.2.2- Immunocytochemistry analysis

The result of immunocytochemistry against MHC is a set of microscopic images where myoblasts
are observed unstained (no expression of MHC) and myotubes cytoplasm has acquired a brown
color (with expression of MHC). MHC is not expressed in the nucleus, so they remain mostly
unstained (although unspecific staining may be observed when developing time has been too
long).
This immunohistochemistry will allow us to calculate the percentage of differentiated and fused
cells and number of nuclei per myotube, being all indicative of the degree of differentiation culture.
To calculate these parameters representative photographs have been taken of each well.
Photographs of each conditions will be divided between the members of each PIP group and
student will count:
- The number of uni-nucleated cells (unfused and not expressing MHC)
- The number of differentiated cells expressing MHC (both uni-nucleated and
multinucleated myotubes).
BC Lab Manual 1 -2022-2023 23
 - In the myotubes, the number of nuclei per tube (in discrete groups such as 2-4, 5-9, and
>10).

To do this we will use ImageJ program with the Cell Counter Plugin, which will help us count the cell
groups.

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