The overall goal of this method is to isolate trypanosomes expressing a particular variant surface glycoprotein or VSG from the rest of the population and identify parasites that have switched from the starting dominant VSG to any other VSG. This method can help answer key questions in trypanosome biology, such as, what are the factors or events that influence VSG switching in a population of parasites. The main advantage to this technique is that it's faster than previously published methods and allows for easy quantification of switch to non-switch cells.
When the lab first started working on antigenic variation, the first problem was how to detect rare switchers. This kind of problem had actually been solved in immunology a number of years ago, through the use of magnetic-activated cell sorting to enrich cells that were negative for a particular trait. Porting non-selection process to antigenic variation seemed like a really promising idea.
To begin, Lister 427 strain bloodstream trypanosomes should be grown to a density of 0.5 to 100, 000 per milliliter. Aliquot a 50 times 10 to the six cell sample into a 15 milliliter tube and then centrifuge. Save one times 10 to the six cells and culture to be used later as positive and negative antibody controls.
Pipette off most of the supernatant leaving roughly 750 microliters in the tube. Transfer the cells to a 1.5 milliliters centrifuge tube. Finally, centrifuge the cells at four degrees Celsius for four minutes at 2800 times g.
And then pipette off all of the supernatant. Resuspend cells in 150 microliters of culture medium, plus primary anti variant surface glycoprotein antibody or anti VSG at the appropriate dilution. Next, vortex the cells at four degrees Celsius for 10 minutes.
Wash with 800 to 1000 microliters of cold culture medium. Centrifuge the cells at four degrees Celsius for four minutes at 5200 times g. After aspirating the supernatant, wash the cells with one milliliter of culture medium and centrifuge again.
Remove the supernatant and resuspend the pellet in 100 microliters of culture medium. Add 110 microliters of magnetic-activated cell sorting microbeads of the appropriate type for the primary antibody. Mix well by pipetting up and down and then vortex.
While cells are vortexing, set up a magnetic-activated separation column. Place a 15 milliliter tube underneath it to collect the priming medium. Add two milliliters of culture medium to prime the column.
Next, remove the sample from the vortex and add 800 to 1000 microliters of cold culture medium. Centrifuge the sample and then remove the supernatant to remove unbound antibody. Wash the pelleted cells with one milliliter of culture medium and centrifuge again.
This time aspirate the supernatant but leave 100 microliters in the tube. Flick the centrifuge tube vigorously until the pellet is fully resuspended. Add one milliliter of culture medium and pipette up and down to fully resuspend the cells.
Remove the 15 millimeter conical tube used to collect the priming medium and replace it with a new 15 milliliter conical to collect the flow-through containing the switched parasites. Apply the cell suspension to the primed column. Collect the flow-through in a 15 milliliter tube below which typically takes six to seven minutes for one milliliter of medium plus trypanosomes.
Next, wash the column two times with one milliliter of culture medium and collect the flow-through in the same tube. Divide the flow-through which should total roughly three milliliters into two 1.5 milliliter microcentrifuge tubes. Then, place the tubes on ice.
Add three milliliters of culture medium to the column and plunge the column into a 15 milliliter centrifuge tube. This will obtain the cells that express the starting dominant VSG. Remove 300 microliters of this eluted material and keep it on ice, the rest can be discarded.
To create a positive control, take approximately 100, 000 cells from the starting culture of cells and pipette them into a 1.5 milliliter microcentrifuge tube. Repeat this to create a negative control. Centrifuge the flow-through samples and positive control sample.
And then remove and discard the supernatant. Resuspend the positive control cells in 100 Microlitres of culture medium with primary fluorophore-labeled anti VSG antibody at the appropriate dilution. Next, resuspend one flow-through pellet in 100 microliters of culture medium with primary fluorophore-labeled anti VSG antibody at the appropriate dilution.
Use the suspension to then resuspend the pellet in the second flow-through tube resulting in one pool tube. Mix well by pipetting up and down. Vortex the cells and then add 800 to 1000 microliters of cold culture medium.
Centrifuge the samples at four degrees Celsius for four minutes at 5200 times g and then discard the supernatant to remove any unbound antibody. Wash the cells with one milliliter of culture medium, then centrifuge the cells. The positive and negative controls and the eluted cells previously stored on ice.
Discard the supernatant. After that, add 148.5 microlitres of culture medium to the samples. Next, add 25 microliters of absolute counting beads, followed by 1.5 microliters of Propidium Iodide staining solution.
Resuspend the positive and negative control pellets with 175 microliters of culture medium. Samples can now be run on the flow cytometer. These flow cytometry plots, show results of cell separation from populations of trypanosomes induced to create a double-strand DNA break versus uninduced cells.
The left hand panels show the forward versus side scatter, which allows elucidation of the live versus dead cells and beads added to the sample. Gates can then be generated that determine these based on the scatter characteristics. The right hand panels show only those cells that could be classified as live based on the scatter gates generated in the left hand panels.
The dominant starting VSG in this case was VSG two. Cells that stain positively for Propidium Iodide are dead cells. And these are seen in the Q one and Q two regions of the plot.
Cells that stain positively for the dominant VSG and negatively for PI fall into the Q three region and are contaminants. Finally, cells in Q four are live cells that stain negatively for the dominant VSG which are considered switchers. This plot shows the calculated observed switching frequencies of the trypanosomes populations with versus without induced breaks.
The level of stochastic switching in vitro is fairly low. While inducing a break results in significantly more cells, switching to the non-dominant VSG. Once mastered, this technique can be performed in about three hours.
while attempting this procedure, it's important to keep samples cold throughout and to adequately resuspend pellets especially prior to isolation on the column. Following this procedure, other methods like RNA sequencing can be used to answer additional questions like what VSGs are being expressed in a switch population. Those method is typically used to measure VSG switching, it can also be used for the isolation and quantification of parasites expressing any surface protein of interest.
