Friday, August 17, 2007

Week 8 Attachment Sharing

Hey wait! Don’t switch to another blog. One of my colleagues gave me a unique and interesting exam question given in a university. If one day you become a lecturer, you should set such questions. Go read the 1st comment for this post!

Ok..now the boring part..

This few weeks I have learnt quite a lot of new stuff like flow cytometry analysis, plasmid purification, gel retardation assay and also confocal fluorescence microscope(still learning). However, I will focus on flow cytometry in this week post because I think it is more interesting.

Introduction

In this experiment, cancer cells are tranfected with polymer-DNA complexes. Since naked DNA/plasmid cannot be efficiently delivered into the nucleus of the cells, my lab designs carriers such as cationic polymers to deliver the DNA into the cells. In this case, the DNA is an EGFP encoded plasmid, meaning, cells that receive the plasmid are able to express the green fluorescence protein(GFP). Such DNAs are also termed reporter genes. To measure how good the carriers are in delivering the plasmid, we measure the level of gene expression.

Purpose of flow cytometry in this experiment

Flow cytometer is used to measure the gene expression. Cells tranfected with the EGFP plasmid will fluoresce green when exposed to blue light. The unique factor of flow cytometer is that it can measure the fluorescence per cell, hence you can know how many cells are expressing the gene. Unlike normal spectrophotometers which measure the transmission of light in a bulk of sample.

Flow cytometers can measure more that 1 type of fluorescence simultaneously per cell and also measure different types of cell. However in this experiment, the cell type used is HEK 293(human embryonic kidney cells) and the fluorochrome is green fluorescence protein(GFP). The brand of the flow cytometer used is BD LSR II( 3-laser FACS analyzer).

Before, using the machine, cells must be prepared for analysis. Single cell suspensions must be obtained. Though the process is simple, but it is very tedious and time consuming. I don’t see the significance of explaining individual steps, so if you are interested, just send me a comment.

I usually run 3 replicates for each condition(different concentration of polymer-DNA complexes added into cells). In this experiment, I have 10 condition, hence, I will have 10x3=30 samples. The whole process can take a day! I’m always super exhausted at the end of the day.

Principle of flow cytometry


Image 1: taken from http://biology.berkeley.edu/crl/flow_cytometry_basic.html

When measuring, cells will flow into a stream of fluid in a flow chamber, allowing only single cells to pass due to the reduced diameter. Lasers are often used as light source in flow cytometry. In this case, argon ion laser is used to excite the GFP cells at 488nm. The corresponding fluorescence signals are picked up by optical detectors at 503-530nm.
The signals are then ‘processed’ further, but it is so technical, so no need to explain as the machine does everything. =))

Data presentation

This is the most important part. A computer is connected to the flow cytometer to analyze the signals. It can be presented in many ways, but I only use two, histograms and dot plots.

1) Dot plots( 2 dimension)

Image 2

y-axis: SSC-A(Side SCatter): parameter that relates to the density/granulariy of the cell.
X-axis: FSC-A (Forward SCatter): parameter that relates to the size of the cell.

Both axes allow us to create a 2 dimensional plot.

Dot plots are often used to measure cell size and density. Based on both axes, it will position the cell in a form of ‘dot’. Each dot represents 1 cell. Only cells(dots) that are in the scatter gate are analyzed, while the others are ignored. The scatter gate is created by the user(me =)). Hence, it can be shifted to the right or left, or even make it larger. So how do I decide? I use controls and all my other samples are based on that.

Why do I need controls?

Reason: To properly set the conditions for flow cytometry, negative (untreated cells: no GFP) and positive controls are required for each cell type and for each fluorescence dye. Different cell types auto-fluoresce at different intensities, so a negative control for one cell type might appear positive for another cell type. In addition, if one cell type is significantly larger or more complex than the other, their forward- and side-scatter settings will differ.

In real life, dot plots are often used in hematology to distinguish different lineages of blood cells, as different type of cells will appear at different parts of the plot based on their density and size.

2) Histogram(single dimension)

Image 3

y-axis: count: no. of cells
x-axis: GFP-A: relative fluorescence intensity

This is the simplest way to present the data. Peaks that are in the right ‘box’ are GFP positive, while those in the left are non-GFP cells. Again, I must have negative and positive controls to ‘gate’ the peaks.

Finally, The flow cytometer will automatically tell me how many GFP positive cells based on the data analysed and the scatter gate I chose.

Image 4: Interpretation of data

Note: Image 2-4 are posted with permission.

Till here then.

Nisha

0503254E

TG02

15 comments:

royal physicians said...

SCIENCE + LOGIC= A grade!!
The following is an actual question given on a University of Washington chemistry mid term.

Bonus Question: Is Hell exothermic (gives off heat) or endothermic (absorbs heat)?

Most of the students wrote proofs of their beliefs using Boyle's Law (gas cools when it expands and heats when it is compressed) or some variant.

One student, however, wrote the following:

First, we need to know how the mass of Hell is changing in time. So we need to know the rate at which souls are moving into Hell and the rate at which they are leaving. I think that we can safely assume that once a soul gets to Hell, it will not leave. Therefore, no souls are leaving. As for how many souls are entering Hell, let's look at the different religions that exist in the world today.

Most of these religions state that if you are not a member of their religion, you will go to Hell. Since there is more than one of these religions and s! ince people do not belong to more than one religion, we can project that all souls go to Hell. With birth and death rates as they are, we can expect the number of souls in Hell to increase exponentially. Now, we look at the rate of change of the volume in Hell because Boyle's Law states that in order for the temperature and pressure in Hell to stay the same, the volume of Hell has to expand proportionately as souls are added.

This gives two possibilities:

1. If Hell is expanding at a slower rate than the rate at which souls enter Hell, then the temperature and pressure in Hell will increase until all Hell breaks loose.

2. If Hell is expanding at a rate faster than the increase of souls in Hell,then the temperature and pressure will drop until Hell freezes over.

So which is it?


The Student gets an A+ !!!!!!!!

Nisha
BMT
TG02

J.A.M.M.Y.S said...

Nisha!!

Omg! The student really wrote that? Interesting.. Ok, now back to the question. You mentioned that you usually run 3 replicates right. What do you do when there is an outlier? Because we also do 3 replicates for our experiment, n sometimes we will not get consistent results and we do not know the reason why.

Ming Boon
Tg01

Star team said...

HI Nisha
Very interesting story..anyway, u said that both histograms and dot plots are used..why can't we just use 1 type? & what are the other ways of presentation for the analysis of the signals?
Thanks =)

Eugene Wong
TG02

MedBankers said...

Hey nisha, interesting question there...swear I heard it before. Student is really quite brazen.

Here's my take on it. Given that it is hungry ghost festival where the gates of hell open up (to chinese ghost only, i guess, since other religions don't have this) Hell would freeze over because 1. there are alot of chinese, and everyone goes to hell for some sort of crime (i think, but bear in mind that was what i saw at har pa villa like 10 years ago.), so therefore, more souls will be leaving at a certain rate, leaving hell to expand faster than souls going in.
Of course, this is only once a year, so hell is endothermic and only freezes over once a year. After which, with the sudden rush of souls back the next day, hell breaks loose. Or becomes exothermic.
No offense meant to anyone reading this...and uh, do i get an A too for my spirited attempt?

Anyways, back to the topic.
In event that there is a false positive result in the negative control, do you reset the parameters or can the computer use the results to calibrate the actual results?

- Debra, TG02

royal physicians said...

Hey Ming Boon..

Sometimes i do get an outlier, but it doesn't deviate that much from the other two. Since, i need to plot graph, so my error bar in the graph will be large. But other than EGFP experiment, I will always have 4 or more replicates, so that if there is an outlier, i will just delete it off. I only do 3 replicates for EGFP bcoz very tedious process..hehe

royal physicians said...

Hey Eugene,

hmm..it depends on the purpose of doing the flow cytometry. For example, if you have two type of lymphocytes, you will prefer to use the dot plot because u can see 2 areas of dots. Maybe the top dots in the graph is lymphocyte A, and at the bottom is lymphocyte B.

In my case, i will rather use the histogram, because i'm only interested in looking the level of gene expression, which is proportional to the signal intensity. But i also use dot plots(not necessary actually) to see if got any dead cells or clumps. But, people usually use both graphs to present their data. =))

royal physicians said...

Hey Debra!

haha..ur take is even more interesting! and funny seh, I think we must ask the guard of hell how the temperature of hell is maintained. haha lol.

Ok, regarding ur ques, all my negative controls have confirm no GFP encoded plasmids added, because i left the cells untreated with anything.
Again, i have 3 replicates for my negative control too, and all gave me the same result, if one give me different value(so far never), means something is wrong with the samples, maybe i accidentally add DNA into the cells..hehe =))

Anyway the machine or computer very stupid, they just listen to the user's instruction..hehe

we are the XiaoBianTai-7! said...

Hey nissha nek nek,
Is the green fluorescence protein(GFP)a Mab?

Thanks!
Take care,
Charmaine Tan TG01

we are the XiaoBianTai-7! said...

hello~ i'm using the flow cytometry for my major project too.. heard from my senior that her evaluator was Dr Lim or Mr Paul Chong the other time.. erm.. both are pros in this area.. so it's good to noe the flow inside out.. pray hard our evaluator not them.. anyway.. will be posting some info on flow cyto soon.. hope to share some knowledge with you then.. take care ~

Joan
TG01

royal physicians said...

Hey charmaine!

wei, im not nenek ok, hmph! anyway, its not an mab, it is just some protein that can fluoresce when exposed to blue light, and often used as a fluorescent indicator in biology. yup~~

royal physicians said...

hey Joan..

great! urs is the alvin poh project izit? hmm..tell me wait ya!

Vino said...

hi NISHA!!!
hope ur doing great.. anyway u mentioned that Only cells(dots) that are in the scatter gate are analyzed. is there a certain number of cells or range that have to be achieved or smth??

Vinodhini
TGO2

royal physicians said...

Hey Vino!

hmm..in total, whether or not within the scatter gate, there is a fixed no. of cells measured, which is 10 000 cells(look at image 4). however, once selecting the scatter gate, the no. of cells will confirm change, as I only want those in the scatter gate oni ma.. =))

royal physicians said...
This comment has been removed by the author.
royal physicians said...
This comment has been removed by the author.