BIOSYNTHESIS

OF
MACROMOLECULES
Jess Blanco Piar
2nd Biochemistry Degree

FACS: Fluorescence-Activated Cell Sorting, applied in flow cytometry

In a certain experiment, it is always essential to determine some standard conditions
under it will be conducted. If we are working with cell cultures, one important factor is
the synchronicity of the culture. If you dont synchronize the culture theres going to be
cells in different stages of the cell cycle. Before achieving this, its essential to
characterize the culture, including How are the cells, How are they growing, etc
including the presence of potential pollutants: Sometimes it can be done at first sight,
as in bacterial contamination the excretion of organic compounds induces changes in
pH, which can be measured with a colour indicator.
One important factor of a cells culture is the confluency. It is a measure of the number
of the cells in a cell culture dish or a flask, and refers to the coverage of the dish or the
flask by the cells. For example, 100 percent confluency means the dish is completely
covered by the cells, and therefore growth is inhibited by contact; whereas 50 percent
confluency means roughly half of the dish is covered and there is still room for cells to
grow. The confluency is determined by observing the culture under the inverted
microscope and it can be seen as an ambiguous parameter, since it is determined
solely by the scientist judgment. Despite this, confluency is a very useful parameter
because there is a direct relationship between the percentage of confluency and the
number of cells in a particular stage of the cell cycle.
To determine in which phase of the cell cycle each cell is, one have to measure the
total quantity of DNA. To do this, DNA is labelled with a fluorochrome (Iodide
propidium) and a standard curve is made measuring the DNA quantity versus
fluorescence intensity. Therefore, DNA determination is only effective within a certain
range.
In the FACS, the cell passes through the capillary of the cytometer, which measures
the fluorescence emission of each cell and correlates:
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G0-G1 Phase 2n

S Phase 2n-4n

G2n Phase 4n

Iodide propidium is a large molecule which is incapable of going through the membrane, so
we have to make small holes in it. The problem is that we have to dissolve the membrane a
bit but carefully trying not to break the entire cell. The technic consists in dehydrating the
outer and nuclear membranes of the cell and it is faster that isolate nucleus
-

Treatment with Formaldehyde to denaturalize DNA

Cold-Dehydration with Ethanol

Normal cells grow in a single layer, adhered to the surface, but tumorous cells there is no
contact inhibition and many more layers accumulate. We are working with VeRo cells,
which are established cell lines, cell cultures that will proliferate indefinitely given
appropriate medium and space. This is accomplished by genetic modification.
These cells stick to the surface of the plate using proteins of the extracellular matrix, so we
have to hydrolyze these proteins using trypsin to detach the cells from the plate.
1.- See confluency under the microscope.
2.- Throw up the medium and wash with PBS: We have to wash because trypsin works at
neutral or slightly alkaline pH and the medium is acidified. We use PBS, which is an
isotonic buffered saline solution. It has to maintain an osmotic pressure and the pH. We
cant use distilled water since the cells would swell and break.
3.- Use trypsin to detach the cells.
4.- Watch under the microscope again. The cells have to adopt an spherical shape and
they should move when the culture is agitated, although it is not necessary to separate all
of them.
5.- Separate the cells from the trypsin. We take the liquid and put it in an Eppendorf.
6.- Centrifuge. Discard the supernatant with the trypsin. The last drop can be eliminated
with filter paper. Now we have the pellet with the cells.
7.- Resuspend the pellet. Add formaldehyde to dehydrate for the first time.
8.- Incubate in ice at 4C (15 minutes).
9.- Separate the cells from the formaldehyde. We centrifuge and discard the supernatant.
10.- Second dehydration. We first resuspend and then add the ethanol. We have to add it
drop by drop while agitating in the vortex in order to avoid membrane breakage.
11.- Store the sample at 4C until the next day.
Cells will tend to form glomeruli and to adhere to each other and in the cytometer they have
to pass through the capillary one by one, so we have to break these glomeruli. To do that
we take the sample with an insulin syringe. The cells will pass through the syringes
capillary and they will separate from each other.

RESULTS
This is the graphic we obtained before the FACS went through. The two peaks correspond
with the percentage of cells in each stage. Our values, with their corresponding errors, are
represented by the black line of the graphic. When we make the more-likely statistical
model of our data, we eliminate the background noise obtaining the statistically most
probable values, which are represented in red, and this is the graphic we work on.

File analyzed: CICLO PRACTICAS 04-04-2014_CYJ.fcs

Date analyzed: 4-Apr-2014
Analysis type: Manual analysis
Diploid: 100.00 %
Dip G1: 66.87 % at 49.70
Dip G2: 23.41 % at 99.41
Dip S: 9.71 % G2/G1: 2.00
%CV: 2.47
Total S-Phase: 9.71 %
Modeled events: 4889
All cycle events: 4889
From the former data we can deduce:

Diploid: 100.00 %: 100% of the cells are diploid.

Dip G1: 66.87 % at 49.70: 66.87% of the cells are in G1 phase. Besides, we can
observe in the graphic a maximum of DNA quantity of 49.7, which corresponds with
the maximum of the first peak of the graphic. We can also observe that the
approximated amount of cells is 580.

Dip G2: 23.41 % at 99.41: 23,41% of the cells are in G2 phase, and in this case the
maximum of DNA quantity is 99,41, with an approximated number of cells of 110.

Dip S: 9.71 %: 9.71 % of the cells are in S phase. This is due to the high
confluency that we observed at the beginning of the lesson, approximately a 90%.
This high value of confluency means that cells (which are in a single layer) keep
contacts with each other and therefore inhibiting cell division.

G2/G1: 2.00: The maximum DNA quantity in G2 phase is twice the maximum in G1
phase, due to the fact that in G2 phase the genetic material has been replicated in
order to permit cell division, giving two daughter cells whose DNA quantity is similar
to the one in the resting cell.

%CV: 2.47: Variation Coefficient, give us information about the accuracy in the
practice performance. For the errors not to be of significance the CV has to be less
than 7. We can see that our CV value is relatively small, so the practice is realized
correctly.