The cell suspension was then transferred to a 15 ml centrifuge tube and slowly mixed with 5 ml of warmed culture medium.
This mixture was later added to 200 ml of warmed (preheated) neem oil inside a 500-ml conical flask and placed on a magnetic stirrer with heater, continuously stirred, for 1 h for the acid transesterification to take place.
Animals were permitted to survive for 2 days after the injections, and were overdose-anesthetized prior to transcardial perfusion with 500 ml of warmed (37°C) physiological saline followed by 1,500 ml (for cats) or 2,500 ml (for macaques) of fixative containing 1% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer (PB), pH 7.4 and then by 1,000 ml PB, pH 7.4.
BAL was performed by sequential instillation and aspirations of three aliquots of 60 mL of warmed sterile 0.9% saline.
Mouse mesothelial cells were harvested by injection of 10 mL of warmed 0.25 % Trypsin/EDTA solution into the peritoneal cavity [ 26].
Bronchoalveolar lavage (BAL) was performed by sequential instillation and aspirations of three aliquots of 60 ml of warmed sterile 0·9% saline.
The ovarian cortex biopsy was put in a volume of 2.5 mL of warmed and preincubated supplemented DMEM/F-12 culture medium (composition described below).
Spores were ground in pre-chilled mortars; 200 ml of warmed (37°C) extraction buffer (50 mM Tris pH 8.0, 200 mM NaCl, 0.2 mM EDTA, 0.5% sodium dodecyl sulfate, 0.1 mg/ml Proteinase K, 0.1 mg/ml glycogen) was added.
Therefore, each rat was administered s.c. 5 ml of warmed lactated ringer's solution (Ringer-Lactat nach Hartmann B. Braun, B. Braun, Melsungen, Germany) one hour after induction of anaesthesia.
Campylobacter species were isolated by the addition of 10 mL of the water sample to 90 mL of warmed Campylobacter enrichment broth (product CM0983, Oxoid Ltd ,Basingstoke, UK) and incubated at 37°C for 24 hours, followed by incubation at 42°C for 24 hours.
