In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

The overall goal of this procedure is to measure parat and mezo invasion in a mouse model of malaria infection. This is accomplished by first collecting blood from uninfected animals. The red blood cells are then labeled with fluorescent tags and injected into infected animals.

Blood samples are then harvested from the infected mice and labeled with antibodies against the appropriate cell surface markers of interest. Ultimately, the number of uninfected cells that have been invaded by plasmodium shabo meite, as well as the overall parit of the infected animals can be measured by flow cytometric analysis. So the main advantage of this technique over existing methods for the measurement of meite invasion and para is that the assay is conducted in vivo, thereby allowing for factors such as the influence of the immune system.

So this method can address some key questions in the field of malaria, such as what are the specific elements involved in the MI invasion of red blood cells Before beginning the assay obtain six to eight mice infected with plasmodium shabo at two to 15%Parsit about three hours before the transfusion. Collect 200 microliters of heparinized blood per mouse to be injected from uninfected animals by cardiac puncture. Split the blood into two equal aliquots for the treated and untreated samples and place the tubes on ice.

Next, further, split the samples into two more equal aliquots each. Then add an equal volume of freshly diluted two XAO 6 33 N hs solution to one tube and an equal volume of the two x biotin NHS to the other for both the treated and untreated sample groups. Then immediately mix the tubes and incubate the blood at four degrees Celsius with mixing every 10 to 15 minutes after one hour.

Wash the labeled blood three times with at least two volumes of mouse ringer solutions, supplemented with BSA per wash after the third wash. Spin down the cells one last time without wash solution and then combine the pelleted samples as illustrated. Adding mouse ringer solution to a final volume of 200 microliters of solution per mouse to be injected.

Then at the peak of the parasite S zony, warm the plasmodium shabo infected animals under a heat lamp. After five to 10 minutes, place the mice individually in a restraining device and intravascularly inject 200 microliters of the labeled blood via the tail vein at the appropriate experimental endpoints. Prepare the staining solution by adding JC one to 37 degrees Celsius warmed mouse ringer solution.

Supplemented with BSA while continuously vortexing the solution to reduce the formation of JC one aggregates. Next, add the appropriate antibodies to the solution. Then collect at least one drop of tail blood per mouse into a whey boat, and immediately pipette three microliters of each tail blood sample into 50 microliters of staining solution at 37 degrees Celsius.

After 20 minutes, add 500 microliters of the appropriate freshly defrosted heart solution to the samples for a 20 minute room temperature incubation. Then spin down the cells, discard the supernatant, and add 700 microliters of mouse ringer solutions supplemented with BSA To each sample just prior to analyzing the cells, filter each sample through a 35 micron cell strainer, rinsing the strainer with distilled water between each tube. Then recording at least 500 events for the smallest population.

Select the whole cells, excluding the noise, debris, and platelets based on the forward by side scatter properties of the samples. Next, select the single cells based on either the trigger pulse width or by using the forward scatter peak area to height ratio. Then use the negative fluorescence in the per CPE floor seven 10 and A PCE floor seven 80 channels.

To further select the mature red blood cells and to exclude the red blood cell progenitors and leukocytes for the labeled red blood cell experiment, select each population of labeled red blood cells based on their PE size seven and A oh 6 33 expression, and analyze these groups separately. Finally, select the parasitized red blood cells based on their positive expression of both JC one and herst. In malaria infected animals, the dual positive population represents the parasitized red blood cells while the remaining populations are uninfected red blood cells.

How will Jolie body containing red blood cells or unknown cells? Typically hers positive JC one negative red blood cells account for 0.3 to 0.9%of mature red blood cells making the addition of the JC one dye Critical for the measurement of low parsit. In uninfected samples, less than 0.007%of events should be dual positive depending on the cleanliness of the flow cytometer, which may vary considerably after injecting labeled red blood cells into infected mice.

The a O 6 33 biotin and unlabeled red blood cell populations should be analyzed separately with a parsit of each group determined according to their hooks and JC one fluorescence. These data can then be used to determine the relative invasion rates into the two labeled populations. So following this procedure, Zoya invasion efficiency can be compared between red blood cells of different types in order to discover essential elements to this process.

After watching this video, you should have a good understanding of how to quantify para in the most model of malaria infection, and of how to compare me invasion efficiency in vivo by cytometry.