Monday, 10 May 2010

GGS LIVE - Flow cytometry

Yo all,

welcome again in the Biochemistry Method section. Today we will cover a Flow Cytometry Technique.

Method: Flow cytometry

About: simply saying flow cytometry is a technique where properties of microscopic particles (such as cells or chromosomes) is examined. Flow cytometry is a very powerful method because it can measure many particles in the same time (up to thousands). Additionally, flow cytometry analysis is multiparametric, so different properties of particles can be measured at once.

What: Analysis of cell cycles distribution in different populations of cells.

How: By flow cytometry.

Ok, so let's start with some short background on how the flow cytometer looks like (see picture below)

and how it works ... (what is inside? :) picture below)

Of course this is a simplified scheme:). What you can see on the scheme is:
- a tube with your sample (in our case cells are the particles examined),
- sample collector,
- source of liquids,
- mirrors,
- laser,
- detectors,
- and waster container.

To simplify how the flow cytometer works we can say that particles are sucked from tube, enter machine flow, where they are exposed to laser and finish in waste container. Depending on particles they either adsorb or alter path of the light. This is monitored by different detectors installed in the flow cytometer. Two most important are:
- forward scatter detector - which is placed in line with the light beam and gives us information about the size of cells (particles),
- side scatter - which is placed perpendicularly to light beam and gives us information about surface of cells (particles) for example their roughness etc.
Additional detectors are installed in the flow cytometer which are able to detect other features of cells/particles (for example they can measure fluorescnce of a chemical compund bound to cell membrane giving us idea how many cells do contain such modified membrane).

In our case study we will look at cell DNA content. Ok let's start. To perform flow cytometry we need to first prepare cells. Our experimental design is as follow:
- one control sample (untreated cells, also called unsynchronous population),
- and three treated samples:
  1. nocodazole treated cells (nocodazole is microtubule depolymerizing agent causing cells to stall at the G2/M boarded). For the cell cycle tutorial please visit this post Theory is fundamental - Cell cycle.
  2. hydroxy urea treated cells (HU- hydroxy urea ribonucleotide reductase inhibitor. Inhibition of this enzyme leads to depletion of DNA synthesis substrates - deoxyribonucleotides). This drug stalls cells in or before S-phase.
  3. Drug X treated cells.
First cells are treated with drugs for a specif time or left untreated. After incubation cells have to be fixed. Usually ethanol is used for that purpose but that may be different from protocol to protocol. After fixation all samples are treated with DNA intercalator Propidium Iodide (PI), a red fluorescent dye that stain DNA (see picture below).

After short treatment (when complex between DNA and dye is formed) cells are ready for analysis. So, we simply kick in machine, install our samples and run analysis. The control result from our experiment is shown on picture below.

The left-hand plot is a representation of our population (below you can see it as indicated with a red area), where each spot is a single event (cell or particle). The X-axis is a forward scatter (FSC), which if you remember tells us about the size of particle. If we move along it we go from the smallest cells (G1 phase cells), through S-phase, to the biggest G2 and mitotic cells. The Y-axis is a side scatter (SSC) and if you remeber it tells us about the sufrace of particle/cell. As you can imagine cells in G1 phase have different morphology than cells in S-, G2 or M-phase (that's why they have different position on Y-axis). We can alter position of our population on the dot plot using specific parameters but usually we try to place it in diagonal of dot plot box and between value 200 and 400 on X-axis.
If you have a look at the histogram plot now, you can see that on X-axis we have FL2 parameter (which is actually a fluorescence of our DNA dye) and on Y-axis counts. This plot simply tells us how many cells contains how much of fluorescence. You can imagine that cell in G1 phase, where there is a single copy of DNA, will have smaller fluorescence than a G2 cell which contains doubled amount of DNA. G1 peak is placed at 200 and G2 peak at 400. Everything in between is S-phase cells which contain amount of DNA between 1n and 2n. From this you can see that the most number of cells are in G1 phase then in S- and in G2-phase.

Now let's have a look at flow cytometry results of our treated cells.

We have already covered control experiment, so we start from Nocodazole treated sample.
Nocodazole stops cells at G2/M boarded. You can easily see that dot plot and histogram plot shifted to the right. The block of cells might not be so clear at dot plot but from histogram we can tell that all cells have been stalled in G2 phase (peak at 400). There is no G1-peak or S-phase area.
Hydroxyurea which prevents cells to enter S-phase, decreasaed number of S-phase and G2/M cells. G1 peak is now fatter what suggest that most of cells is stalled in G1 phase. This block is not nice as Nocodazole one but I hope you see difference between hydroxyurea treated and control cells.
Drug X treatment is toxic to cells. We can deffinitely say that it dimnishes S-phase cells and block them in G1. Additionally you can see a small sub-G1 peak which is actually a indication of apoptotic cells (during apoptosis- a programmed cell death, DNA of cell is fragmented and distributed to apoptotic bodies. This is why it appears as smaller, less than 1n).

This is it:) I hope you enjoy it.


1 comment:

  1. I need your permission to use the the flow cytometry Scheme figure in this site. I need an email to contact with.

    Thank you,