The overall goal of this procedure is to induce and visualize cyst wall formation in Toxoplasma Gandhi. In vitro. This is accomplished by first culturing macrophages and fibroblasts.
And then by infecting these host cells with the parasite stress responses can be induced in te gde eye by shocking fibroblasts with pH or activating the macrophages, which will both lead to cyst wall formation. Finally, fluorescent lectin is added to the cultures which binds to the cyst wall, such that cyst can be visualized with immunofluorescence microscopy. Hi, I am Crystal Tobin from the laboratory of Dr.Laura Noel in the Department of Medical Microbiology and Immunology at the University of Wisconsin Madison.
I'm Angela Pollard, also from the No Lab. Today we'll show you a procedure for Toxoplasma Gandhi cyst wall formation in vitro. We use this procedure in our laboratory to study parasite responses to stress conditions.
So let's get started. Begin by placing a sterile round glass cover slip into the bottom of each well of a 24 well tissue culture plate to induce the conversion of Toxoplasma Gandhi to Brady Zoes in human four skin fibroblasts under conditions of high pH and carbon dioxide starvation. Start with a confluent 150 square centimeter flask of human foreskin fibroblasts under a tissue culture hood.
Rinse the confluent flask of human foreskin fibroblasts two times with one XPBS and then add 2.5 milliliters of 0.025%tripsin EDTA. Incubate the flask at 37 degrees Celsius for five to 10 minutes. After the five to 10 minute incubation, tap the flask several times to release the cells from the surface of the plastic.
Next, add 150 milliliters of HFF medium or supplemented delcos modified eagles medium. And use the medium to rinse the flask and collect the cells. Dispense one milliliter of cells into each well of the 24 well plate with cover slips allow the cells to reach con fluency at 37 degrees Celsius in 5%carbon dioxide.
The cells will then be ready to infect with the Toxoplasma Gandhi parasite. Under the tissue culture hood infect a 25 square centimeter flask of confluent human four skin fibroblasts with two times 10 to the six parasites and incubate at 37 degrees Celsius until the cells begin to lice. About two to three days uses cell scraper to remove the infected foreskin fibroblast monolayer from the tissue culture flask.
Then release the parasites from the host cells by passing the dislodged monolayer through a 27 gauge needle. Use a hemo cytometer to determine the number of parasites in the medium and then infect 10 to the fifth parasites into each of the 24 wells of the confluent human foreskin fibroblasts. Previously prepared.
Incubate the cells for three hours at 37 degrees Celsius and 5%carbon dioxide to allow the parasites to invade the host cells. Proceed to induce the conversion of parasites into the Brady Zeolite stage. Remove the DMEM from the infected human foreskin fibroblasts.
Rinse them with one XPBS and then add one milliliter of development medium. An RPMI based medium without bicarbonate adjusted to pH eight. Incubate the cells for three days at 37 degrees Celsius in ambient air.
The cells are now ready to analyze for the presence of Brady zos. The presence of Brady ZOS is determined using the fluorescently labeled lectin docos by fluorescent glutenin or DBA, which binds to the Toxoplasma Gandhi is assist wall to prepare the infected cells for fluorescent imaging. Rinse the wells three times with one XPBS.
Next, fix the cell monolayers with 200 microliters of 3%formaldehyde for 20 minutes. Remove the fixative and rinse the wells with one XPBS. Add 200 microliters of 0.1 molar glycine to the wells and allow them to sit for five minutes.
Remove the glycine and rinse the wells with one XPBS to block and perme the cells. Add 250 microliters of blocking solution. Incubate the cells for 30 minutes.
Remove the blocking solution and rinse the wells with one XPBS. Add 50 microliters of a one to 250 dilution of DBA, which has been conjugated to rumine to the wells. Cover the plate and shake at room temperature for one hour on a platform shaker.
After the incubation, remove the fluorescently labeled DBA by washing the cover slips three times with 0.2%Triton X 100 in one XPBS for five minutes each on a platform shaker finally mount the cover slips using beta shield mounting medium containing dpi. The stain T Gandhi can be visualized using a fluorescent microscope with a filter appropriate for rot domine at a 100 x magnification. As an alternative method for inducing Brady ZO formation, one can activate infected bone marrow derived macrophages with interferon, gamma and lipopolysaccharide or LPS To begin first prepare L 9 29 conditioned medium for this process.
L 9 29 is a mirroring, an applied fibro sarcoma cell line that secretes macrophage colony stimulating factor after long periods of co fluency. This conditioned medium can be used to develop mouse bone marrow cells into macrophages in cell culture to prepare conditioned medium grow 1 150 square centimeter flask of L 9 2 9 cells to co fluency at 37 degrees Celsius. Then incubate them for an additional seven to nine days until they appear spherical and begin to lift from the surface of the flask.
Collect the medium and any detached cells in a 50 milliliter tube and spin at 420 5G for 10 minutes. The resulting sate is the condition medium 1 150 square centimeter flask of L 9 29 cells will yield 30 milliliters of conditioned medium, which is the volume required to prepare 150 milliliters of BMC medium. Remember to prepare DMEM for BMC with the L 9 29 cm to be used for BMC development.
To view detailed video protocols illustrating the method for isolating bone marrow cells. For mouse femur, please view the following video protocols after harvested BMCs have incubated for five days. In bacteriological petri dishes, add 10 milliliters of BMC medium, which will contain conditioned medium from L 9 2 9 cells to each dish and incubate for two additional days after which the cells will be fully developed.
Mature bone marrow derived macrophages or BMS may be passaged for one to two weeks after they have matured to split the bms, remove the medium and add five milliliters of cold one XPBS incubated four degrees Celsius for 30 minutes until the cells begin to lift. Rinse the cells off the plate with a sterile transfer pipette. Pull the BMS in a 50 milliliter conical tube and pellet the cells at 420 5G for 10 minutes.
Resuspend the pellet in 10 milliliters of BMC medium and then use a hemo cytometer to determine the cell density seed. Two times 10 to the fifth BMS per well. In a 24 well plate containing round blast cover slips on the bottom let the cells adhere overnight before infecting with T Gandhi.
Excess cells can be receded on Petri dishes for later use. To activate the BMS prepare activation medium sonicate LPS in a water bath for two minutes to disrupt any aggregates. Then to BMC medium, add 100 nanograms per milliliter LPS and 100 units per milliliter IFN gamma.
Finally, remove the BMC medium from the wells and replace with one of activation medium. Incubate at 37 degrees Celsius, 5%carbon dioxide for three days. BMCs can be stained as previously shown with foreskin fibroblasts.
In these examples, toxoplasma Gandhi I Bradys are labeled with fluorescent DBA in human foreskin fibroblasts. Under pH stress. The cyst wall is fluorescently labeled as shown in this cross section of an in vitro cyst in dms that have been activated with IFN Gamma and L-P-S-D-V-A.
Staining is also present as shown here on the surface of the vacuole. We've just shown you how to stain in vitro Toxoplasma Gandhi, cyst in fibroblasts and activated macrophages. When doing this procedure, it is important to remember to plan for the parasites and host cells to be ready on the same day.
So that's it. Thanks for watching and good luck with your Experiments.
