Working with Strongyloides ratti

Reverse transkriptase Qiagen Invitrogen

SensiScript SuperScriptII, Super-ScriptIII

RNase A Roche DNase-free RNase

Taq-Polymerase New England Biolabs

Invitrogen

DNA-Polymerase DNA-Polymerase

T4-DNA-Ligase New England Biolabs,

Fermentas

DNA ligation

Trypsin Promega Tryptic digestion for MS

For the separation of the respective stages from the charcoal culture the Baermann method was used (Fig 2.3.1.2-1 B) (Whitehead, 1965). Warm water (35–38°C) was filled in a funnel which was closed at the lower end using a clamp. A steel sieve was placed on top of the funnel.

About ¼ of the steel sieve had to be covered with water. A piece of cotton was laid into the sieve and the charcoal culture was carefully transferred into the cotton. A lamp was placed directly next to the funnel to maintain warm temperatures.

2.3.1.3 Preparation of iL3

For the isolation of iL3 faecal pellets were collected on days 6-16 after subcutaneous infec-tion of male Wistar rats with 1,800–2,500 iL3. Charcoal coprocultures were set up and incubated at 26°C. The culture dishes were incubated 5–7 days for the collection of iL3. For the recovery of iL3 the Baermann method was used as described in the previous section. After separation of iL3 from the charcoal culture the larval suspension was pre-cleaned by rinsing the larvae a few times in a suction filter. It was avoided to completely dry the suction filter. Infective L3 were then washed four times in sterile washing solution.

A B

Figure 2.3.1.2-1 (A) Setup for a charcoal culture dish.

(B) Setup of the Baermann funnel routinely used at the BNI for isolation of S. ratti stages from faecal cultures.

2.3.1.4 Preparation of the free-living stages

For the isolation of free-living stages faecal pellets were collected on days 6-16 after sub-cutaneous infection of male Wistar rats with 1,800–2,500 iL3. Charcoal coprocultures were set up and incubated at 26°C for 24 hours. The following steps were performed as described in the previous section.

2.3.1.5 Preparation of parasitic females

For the recovery of parasitic females (pf) male Wistar rats were infected with 2,500 iL3.

On days six and seven past infection the rats were sacrificed and the small intestine was removed beginning from the stomach and ending approximately 10 cm before the appendix. The intestine was pre-cleaned by emptying the contents through careful squeezing of the intestinal walls. After that the intestine was opened longitudinally using scissors and cut into strips of about 8–10 cm length. The strips were then washed three times by gentle shaking in three different glasses filled with 500 mL of water or PBS to remove residual debris. To separate the females from the tissue the strips were placed directly on the sieve without the cotton in a Baermann apparatus and incu-bated for three hours. After sedimentation of the females 50 mL of the solution in the Baermann funnel were filled in a Falcon tube. After a second sedimentation of the females they were trans-ferred to a 1.5 mL tube using 1 mL pipet tips and washed at least six times in sterile washing solution. Between the washing steps the tube was centrifuged at 1,000 rpm for one minute. Using the repeated centrifugation steps at low speed led to a separation of parasitic females from tissue and residual eggs and first stage larvae. The female suspension was now used for in vitro culture, the preparation of extracts or for the preparation of RNA.

2.3.1.6 Preparation of E/S products

Freshly harvested and washed iL3, pf or free-living stages (fls) were carefully suspended in sterile worm culture medium under the laminar flow hood. The incubation densities were not exceeding 30,000 iL3/mL, 15,000 fls/mL and 100 pf/mL. Depending on the amounts either cul-ture dishes or culcul-ture flasks were used. iL3 were incubated 24 hours and pf 72 hours at 37°C. Fls were incubated at 26°C for 24 hours. After the incubation period vitality and sterility were checked under the microscope. An additional test for sterility was performed by placing 5 µL of

culture medium on blood agar plates and subsequently incubating the plates at 37°C for 24 hours. Only sterile cultures were used for further processing.

For the inhibition of protein synthesis sterile cycloheximide (CHX) and sodium azide stock solution was added directly before the incubation of iL3. CHX was added to a final concentra-tions of 50 and 70 µM. Sodium azide was added to final concentrations of 0.5 and 1.0%. After two hours of incubation the culture medium was removed, an equal amount of new medium was added and the larvae were further incubated for 24 hours.

0.5 M prolyl oligopeptidase inhibitor stock solutions of compounds 1A, 1B, 2A and 2B were prepared in PBS and sterile filtered. The stock solutions were added directly before the incubation of parasitic females at final concentrations between 1–10 mM.

2.3.1.7 Preparation of worm extracts

Freshly harvested worms were washed three times in M9 buffer and twice in homogenisa-tion buffer (HB). After removal of the supernatant the worms were either directly processed fur-ther or frozen at -70°C until use. For furfur-ther processing worms were placed on ice and HB buffer with 2 mM dithiothreitol (DTT), 1x Complete Mini protease inhibitor was added. One steel bead per tube was added and the worms were vortexed for ten minutes, after removal of the bead, sonicated for 30 seconds on ice four times. After spinning at 14,000 g for 20 minutes the super-natant was transferred into a new tube.

2.3.1.8 Whole worm analysis

Frozen larvae were picked individually using a 10–100 µL pipet under the microscope.

Picked individuals were transferred to a clean tube and centrifuged at maximum speed for two minutes. The aqueous phase was discarded carefully using a 10 µL pipet without accidentally taking out any worms. 20 µL lithium dodecyl sulphate (LDS) loading buffer was added to each sample and heated to 95°C for ten minutes. The buffer then was loaded on NuPage^®Novex^®tris acetate mini gels and the electrophoresis performed until the buffer front had moved approxi-mately 2 cm into the gel. The gel was Coomassie stained. After destaining each lane was cut out in either one or more gel strips depending on the number of bands visible. The samples then were reduced, alkylated, subjected to in-gel tryptic digest and loaded onto a mass spectrometer.

Database searches were performed as described in 2.3.4.

2.3.2 Molecular biological methods