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.

Maciek GGSTEAM

Theory is fundamental - Cell cycle

Hello BioFreak Reader,

Welcome in a brand new section where we will try to shortly and nicely cover fundamental topics of biochemistry. We are going to start with a Cycle cycle:)

What is cell cycle - (also known as cell division) is a set of events which main objective is to duplicate genomic material (DNA) equaly distribut it to two doughter cell. Four distinct phases can be distinguished during each cell cycle (see picture below). Those are G1, G2 (known as gap phases), S- and M-phases. S-phase (S stands for synthesis) occurs between gap phases and in this phase DNA duplication occurs. M-phase (M stands for mitosis) is the last phase of cell cycle, where cell finaly divides giving live to new cells. Each phase of cell cycle is extremely important and all phase specific events had to be finished before cell enters proceeds to the next one.

Maciek GGSTEAM

GGS LIVE - broken chromosomes

Welcome you all,

today in Biochemistry Methods section we discuss how to assess chromosomal stability. Chromosome aberration is one of the methods.

Method: Chromosome Aberrations.

About: Chromosome aberration assay is a direct method to assess chromosomal abnormalities.

How: By visual examination of chromosomes.

To examine chromosomal stability of chicken DT40 cell line we first need to prepare chromosomal spreads (please have a quick look at this post Meet the chickens - chook chromosomes at hand). To prepare such chromosome spreads, we have to capture them in metaphase (subphase of cell division - mitosis). Usually drug called colcemid is used for that (look at the picture below).

Colcemid is a drug that interfere with formation of mitotic spindle by depolymerization of microtubules. This way we enrich population of cells that already condensed their DNA, which now has a form of chromosomes.
Later cells are harvested and incubated in hypothonic buffer (what cause their swelling) to make them more fragile. Next step is to fix them in such state and drop them onto slide. Cells burst/break when they are dropped, releasing chromosomes (as on the picture below or in this post Meet the chickens - chook chromosomes at hand).

Picture taken by Kliszczak M.

As you can see, breaks in chromosomes can be easily seen and scored (red arrow heads). Usually cells are left untraeted (to score spontaneous aberrations) and treated with ionizing radiation (IR) or with drug of interest. Chromosomal aberrations are then scored and compared to untreated sample.

I hope you enjoy it.

Maciek GGSTEAM

Meet the chickens - sister chromatid labelling

Yo all,

welcome again in Meet the chickens section. Today a specific labelling of chromosomes (actually sister chromatids) will be presented.

As you probably remember from previous post (Meet the chickens - chook chromosomes at hand), there is a way to examine karyotype (set of chromosomes) of chicken DT40 cell line (or any other cell line:). In this post we will combine this technique with a labelling procedure. This protocol will give us oportunity to see differential staining of sister chromatids.

Ok lets go.

In this protocol a nucleotide derivative, called BrdU (5-bromo deoxyuridine) is used to labell DNA for two cell cycles (see the picture below).

Simply saying, cells are treated with BrdU, which is incorporated to DNA during S-phase (DNA synthesis phase of cell cycle). It is important that labelling is performed for exactly two cell cycles. This is because after two cell cycles (two subsequent S-phases) one of sister chromatids is complately substituted with BrdU where other one is half substituted half not. This feature of labelling can be now used to distinguish between sister chromatids (they simply have different physical and chemical properties).

First cells are stopped in metaphase (cell division phase) using a specific drug (usually colcemid a microtubule depolymerysing agent) that prevents mitotic spindle formation. After that, cells are swollen (to make them fragile) and fixed (to fix their state:). Further, cells are droped onto slide to open them and release chromosome spreads. Next few steps leads to differential staining of sister chromatids (see picture below).

In the first step, cells are treated with a DNA intercalating dye called Hoechst 33258 (this is a fluorophore that adsorbs UVC light at 258nm). Intercalating means that it gets in between the DNA base pairs stacks (see picture below)

Red bars represent an intercalating agent bound to DNA/

Picture taken from http://en.wikipedia.org/wiki/File:DNA_intercalation.jpeg

As you remember one of the chromatids is complately BrdU substituted. BrdU contains bromide and occupy more space than a regular nucleotide (remember that bromide atom is way more bigger than carbon, hydrogen or nitrogen). Additionally BrdU nucleotide is light sensitive. Hoeachst intercalation occurs only in monosubstituted chromatid but not in disubstituted one (there is not enough space for Hoechst intercalation, because of two bromide atoms).

Further, slides are exposed to UVC light. Hoechst 33258 adsorbs the UVC light protecitng monosubstituted chromatid from UV light degradation. Subsequently DNA is stained with another dye called Giemsa stain. Degraded DNA do not stain with Giemsa dye revealing a differential effect as on picture below. Additionally to UVC light effect it is believed that different set of proteins are bound to BrdU disubstituted chromatid comparing to monosubstituted (what leads to differential staining). 

Picture taken by Kliszczak M.

As you can see on the picture above mono- and disubstituted chromatid are indicated with blue and red arrowheads, respectively. Monosubstituted chromatid appears darker (as a result of Giemsa staining) than disubstituted (pale colour, no staining).

This technique is being used to assay chromosomal stability. Any DNA damage that occurs in the cell and is repaired by recombination (exchange of DNA information between the sister chromatids), will be visualized by this technique. You can see such events on the picture above. Two of them are indicated with black arrowheads. Some gene mutations (for example Rad54 or Rad51 - these genes code for proteins involved in DNA recombination pathways) leads to decreased number of Sister Chromatid Exchanges (SCE). Usually a specific drug (in this case Mitomycin C) is used to induced SCE's. After induction SCE's frequencies between Wild Type and mutant strains are comapred.

I hope you enjoy it.

Maciek GGSTEAM

Western Blot in Pictures

Hello Biofreak,

I do not have to explain what this setion is going to be about:)

Enjoy!!

!!To download the WESTERN BLOT TUTORIAL IN PICTURES click on the link!!

MACIEK GGS