Cyst Purification Protocol (Sinai Lab)

Infect CBA/J mice (Jackson Labs) with 200-500 cell culture derived ME49 tachyzoites. We have noted that parasites maintained in culture tend to gain virulence and as a result one can experience anywhere from 10-80% of the mice succumbing to the acute infection. The level of variability is a problem in planning the experiments. This is much less of a problem if infecting with in vivo cysts as below.

Alternatively, you may serial passage cyst containing brain homogenate through IP injection of 18-20 cysts freshly harvested in the brain homogenate from Step 11 of the protocol below following dilution with PBS to have 18-20 cysts per 200 L of brain homogenate per mouse.

Mice that do become symptomatic will exhibit ruffled fur between 7-10 days post infection. These should be monitored as animals that become hunched/ataxic and somewhat unresponsive to gentle prodding are not likely to survive and should be euthanatized. It is possible that mice remain asymptomatic. If asymptomatic for 3 weeks antibody responses should be checked by western blot.

Tissue cysts can be purified from day 21 (week 3) and onward. Cysts were obtained from week 2, but they were small and fragile and resulted in low yields.

Euthanize the mice using CO2 asphyxiation and harvest the brain using sterile equipment. If relevant collect blood by cardiac puncture for serum.

Once the mice are harvested you must proceed throughout the entire purification.

Processing the Brains and purification of cysts using Percoll EquipmentSmall porcelain mortar and pestle (sterilized in an autoclave pouch) Sterile cell scraper (small head) (Corning Incorporated #3010)5ml syringesNeedles:

16 G needles18 G needles20 G needles 23 G needles

Sterile PBS with 1% Tween 80 (filter sterilize)Percoll (Sigma # P1644-100ML)

Percoll Gradient90% Percoll in PBS (5 mL of filter sterilized 10X PBS + 45 mL of Percoll)40% Percoll in PBS (5 mL of filter sterilized 10X PBS + 20 mL of Percoll + 25 mL autoclaved dd H2O)20% Percoll in PBS (5 mL of filter sterilized 10X PBS + 10 mL of Percoll + 35 mL autoclaved dd H2O)

This is a modification of the original Percoll gradient which consisted of 90% and 30% layers (Cornelissen et al., 1981). When doing the first set of gradients you can add a couple of drops of blood to the brains to get a more pronounced RBC band as a marker in the gradient. You may

set up the gradients just prior to loading the brain homogenate, or you may set them up before harvesting the mice. If you set them up before harvesting, leave them in the fridge until ready. Layering the Gradients:1. The gradients should be set up in a standard 50ml Falcon tube.

Directly add 9 mL of the 90% Percoll to the bottom of the tube using a pipette.

2. Measure out 4.5 mL each of 40% Percoll and 20% Percoll into two separate 14 mL round bottom snap caps tubes.

3. Collect the 40% Percoll into a 5 mL syringe with a 16 G needle (Figure 1).

4. Layer the 4.5 mL of 40% Percoll on top of this 90% Percoll cushion by carefully tilting the tube and slowly dispensing the Percoll against the wall of the tube (with the flat opening of the needle against the wall) right above the 90% layer (Figure 2).

5. Layer 4.5 mL of 20% Percoll as above. Be extra careful layering this as the 40% and 20% layers tend to mix easily.

6. Place in fridge until sample is added, or directly add the brain homogenate by layering as in Step 4 if the mice have already been harvested and the brain homogenate is ready.

Homogenization of the Brains (must be done in the hood):Set up:Clear out the hood.Wear PPE (gloves, cuffs, lab coat).Fill a flat, long ice bucket with ice and cool the mortar, pestle, and PBS with 1% Tween 80.Fill a 4 L plastic beaker with 2 L of 10% bleach for decontamination of equipment without overflowing.Have a little surgical table prepared (a Styrofoam lid with aluminum foil covering is sufficient). Place a biohazard bag in the hood to dispose mice in.Have your 5 mL syringes and 16-23 G needles ready, along with the cell scraper.Harvest the mice:We typically house 4 mice in a cage and process a cage at a time. Thus 2 brains are pooled for processing and for each gradient.Do no more than 3 brains per homogenization/percoll gradient as this will overload the gradients and result in a significant loss of yields.

7. Homogenize 2 brains (Figure 3) using a mortar and pestle until you get a consistency of toothpaste (do not add PBS or any buffer at this stage) (Figure 4).

Figure 2

Figure 3 Figure 4

8. Add 3 ml PBS with 1% Tween80, and using the pestle, incorporate the brain paste uniformly by scraping the material off the wall of the mortar and the pestle with the cell scraper as needed (Figure 5). Dispose of scraper into the bleach.

9. With the brain paste incorporated into the PBS-Tween80 still in the mortar, syringe passage the homogenate 5 times using a 5ml syringe and 16 G needle to break up large clumps (Figure 6). Prior to disposal of every needle, draw the 10% bleach solution up into the syringe to decontaminate it and then discard it into the proper sharps container. Repeat this with a fresh syringe and an 18 G needle.

10. With a new syringe, passage homogenate through a 20 G and a 23 G needle around 10 times. If you have not thoroughly broken up large clumps with the 18G/20G needles, they will get stuck in the smaller needle and create a dangerous situation!

11. Add 6 mL PBS (NOT PBS + 1% Tween-80) to the brain homogenate and layer onto the top layer of the Percoll gradient (Figure 7). Do this as above in Step 4 with a syringe so that you cause a minimal disturbance of the interface. Be particularly careful since you are loading on top of the 20% Percoll layer.

12. After placing the gradients into an enclosed container, centrifuge (Allegra 6R Centrifuge) at 2200 rpm for 18 minutes at 4oC. (Note: start timing once speed is reached and set the centrifuge for a high brake when stopping) Use a timer separate from the centrifuge’s timer to be more precise.

13. As the gradient is spinning, you may decontaminate all materials (mortar, pestle, surgical equipment, etc.) into the 10% bleach and remove the plastic beaker from the hood and wash out in the sink. Replace the ice in your bucket with fresh ice and place the bucket back into the hood.

Once spun the gradient will look like Figure 8.

Harvesting the Gradients:

We harvest the gradients using a peristaltic pump (Rainin). A glass capillary tube is attached to each end of the plastic tubing (Fisherbrand # 14-190-510, 1.52 mm with a length of 16”).

Calibration of the Pump:Prior to harvesting the gradient it is necessary to calibrate the pump. To do this, fill 10 mL of PBS into a tube and

Figure 5

Figure 6

Figure 7

Cyst LayerRBC Layer

} Brain Homogenate

Figure 8

insert the capillary at the bottom. Pump out the volume and time how long it takes to fill a fresh tube. Establish the flow rate (it will depend on the tubing you are using and the speed of the pump), at about 2 mL per minute (1 mL every 30 seconds). With the above tubing, a setting of 11.1 on the peristaltic pump is optimal.

Collecting the Gradient:Once calibrated, set 19 labeled Eppendorf tubes in a rack. (You can set up a fraction collector but since one is typically collecting no more than 4 gradients this is not necessary).

14. Carefully lower the capillary to the bottom of the gradient, making sure it is at the bottom and not floating up (Figure 9).

15. Turn on the pump and start the timer when the first drop falls into the first Eppendorf tube. At 30 second intervals, move to the next tube while transferring the filled tube to the ice bucket.

16. All cysts are above the RBC layer, which is typically at fractions 9-10. The cysts are most concentrated in fractions 14-16. Collect around 19 fractions as there is nothing to be gained from collection of the brain homogenate layer which would be around fraction 20.

Counting the Cysts:

17. Add 100 L of PBS to one row of a 96 well plate for each gradient (you may use one 96 well plate for both gradients, skip a row in between).

18. Transfer 10 L of each fraction into a corresponding well. Since there are no cysts below the RBC layer, you may start with fraction 9.

19. Spin the plate at 2000 rpm for 8 minutes.

20. Use an inverted scope with the 10X and 20X objective to count each well for cysts, using the RBCs to focus on the correct plane (Figure 10, red arrows). (Note: Also check the corner of the wells for cysts)

21. Count the number of cysts per well to establish both the number and distribution in the gradient.

Figure 9

Gradient

Flow Direction

Figure 10

Storing the Purified Cysts:

22. Pool the fractions containing cysts into a suitable tube to allow for a minimum of a 5 fold dilution with PBS (3 mL of fractions + 12 mL of PBS in a 15ml conical).

23. Spin at 2000 RPM for 10-15 minutes.

For pelleting the cysts:24. Aspirate off the supernatant, then store at -80 °C.

For imaging the cysts:24. Aspirate off the supernatant, and then calculate for around

300 cysts per 500 L and dilute accordingly in PBS.

25. Prepare the slides to be spun (Figure 11) (slide, filter card, funnel, and metal clamp).

26. Add 500 L into the funnel of each slide (Figure 12).

27. Spin at 750 rpm for 5 minutes at medium acceleration in a Cytospin (Shandon Cytospin 4).

28. Disassemble the slide clamp and discard filter card after removing it carefully (do not slide it off). (Note: Continue to wear gloves as these are live cysts until they are fixed)

29. Store slides in either 3% PFA (store at 4 °C) or ice cold MeOH (store at -20 °C) in a Coplin jar. Those slides that are fixed in 3% PFA should not be stored longer than one week due to higher background levels when staining. Those slides stored in MeOH can be kept much longer without consequence.

Literature Cited

Figure 11

Figure 12

1. Cornelissen, A. W., Overdulve, J. P. & Hoenderboom, J. M. Separation of Isospora (Toxoplasma) gondii cysts and cystozoites from mouse brain tissue by continuous density-gradient centrifugation. Parasitology 83, 103-108 (1981).