Collecting and Processing Saliva

Saliva collections is recommended to be done in the morning (please aim for 10-11am if possible), and ask the subject to refrain from eating, drinking, or oral hygiene procedures for at least 1 hour prior to the collection.

The subject will be given drinking water (bottled) and asked to rinse their mouth out well (without drinking the water).

5 minutes after this oral rinse, the subject will be asked to spit whole saliva (WS) into a 50ml sterile Falcon® tube. The subjects need to refrain talking. It is better for subject to drop down the head and let the saliva run naturally to the front of the mouth, hold for a while and spit into the tube provided. Subjects will spit into the collection tube about once a minute for up to 10 minutes. The goal for each whole saliva donation should be about 5ml. Require that the tube is placed on ice while collecting whole saliva. CROTICAL: Remind the subjects not to cough up mucus as saliva is collected, not phlegm.

COLECTED SAMPLES ARE TO BE KEPT ON ICE AT ALL TIMES PRIOR TO PROCESSING.

Use 2 x 2 cotton gauze to block the opening of each parotid duct; dry up the floor of the mouth and block the openings of the sublingual gland (both sides) and have the subject raise their tongue slightly to elevate the opening to the SM gland. Begin to collect SM saliva by using sterilized Wolf device. A sterilized and disposable yellow tip (for pipette P200) is connected into the device and changed between every collection. During the collection at 2 minute intervals, a few grains of citric acid powder are swabbed with a moistened cotton applicator onto the lateral dorsum of the tongue to stimulate the secretion. Aim to collect at least 200μL SM.

Similar to the protocol described above for SM collection. The only difference is that the ductal orifices of the submandibular gland will be blocked off. Aim to collect > 100μL SL every time.

Use Parotid cup to collect PR. Parotid cups may be placed bilaterally if the clinical investigator so chooses. This will allow for the simultaneous collection from each parotid gland. The citric acid stimulation will be performed as described above. Aim to collect > 1mL PR.

The first 0.1ml of parotid saliva collected will be discarded to insure that fresh parotid saliva is obtained.

To each 100μl saliva, add:

• 0.2μL proteinase inhibitor cocktail from standard stock solution (Sigma, cat# P8340), invert gently.
• 0.3μL Na3OV4 (Sigma, cat# S6508) from standard stock of 400mM, invert gently.

Centrifuge specimens at 2600xg for 15 minutes at 4°C; (if you note that incomplete separation has occurred, you may increase the spin time to 20 minutes). Remove the supernatants from the samples and mark them as plus “super” which stands for the supernatant phase of the saliva. Taking care not to disturb the pellet and please keep the pellet as is in the original tubes, you may like to add marks to the original tubes with “pellet”.

Freeze samples at -80°C.

Saliva collections is recommended to be done in the morning (please aim for 10-11am if possible), and ask the subject to refrain from eating, drinking, or oral hygiene procedures for at least 1 hour prior to the collection.

The subject will be given drinking water (bottled) and asked to rinse their mouth out well (without drinking the water). 5 minutes after this oral rinse, the subject will be asked to spit whole saliva (WS) into a 50ml sterile Falcon® tube. The subjects need to refrain talking. It is better for subject to drop down the head and let the saliva run naturally to the front of the mouth, hold for a while and spit into the tube provided. Subjects will spit into the collection tube about once a minute for up to 10 minutes. The goal for each whole saliva donation should be about 5ml. Require that the tube is placed on ice while collecting whole saliva. CROTICAL: Remind the subjects not to cough up mucus as saliva is collected, not phlegm.

COLECTED SAMPLES ARE TO BE KEPT ON ICE AT ALL TIMES PRIOR TO PROCESSING.