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

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