Submission guide

1. Collect blood via venipuncture using an EDTA (purple-top) or serum separator tube (SST, yellow-top).

2. Gently invert the tube 5-10 times to mix anticoagulant (for plasma samples).

3. Let the blood clot at room temperature for 30 minutes (for serum samples).

4. Centrifuge at 1,500-2,000 g for 10 min at 4°C to separate serum/plasma.

   Note: separate plasma/serum from blood cells as soon as possible.

5. Transfer the supernatant (plasma or serum) into pre-labeled cryovials, avoiding the buffy coat.

6. Immediately store the aliquots at -80°C.

7. Transport samples on dry ice to prevent degradation. Avoid repeated freeze/thaw cycles.

1. Remove media. Carefully aspirate and discard the culture media from the adherent cells without disturbing the cell layer.

2. Wash with 500 µL of 75 mM ammonium bicarbonate (pH 7.4, 37°C). Add a volume of 75 mM ammonium carbonate solution (37°C) equal to the original media volume to the cells, gently swirl the dish to wash the cells, aspirate and discard the ammonium bicarbonate solution. Repeat the washing step.

3. Add ice-cold 80% (v/v) methanol (same volume as the original media) to the cells, immediately scrape the cells using a cell scraper or pipette tip to detach them, transfer the methanol-cell suspension to a centrifuge tube (e.g., 2 mL Eppendorf tube). Repeat the extraction with 80% v/v methanol, scrape again to ensure all cells are collected, and combine this suspension with the first one in the same centrifuge tube.

4. Store the methanol extract at -20°C or colder until shipment.

Note: Ammonium bicarbonate solution can be substituted with PBS.

1. Remove media. Harvest necessary volume of cell suspension, centrifuge cells at a low speed (e.g., 1000 rpm for 5 minutes) to pellet the cells. Carefully aspirate and discard the culture media.

   Note: At least 500,000 cells are needed for most assays.

2. Wash cells. Add a volume of 75 mM ammonium bicarbonate solution (37°C) equal to the original media volume to the cells. Gently pipette up and down or vortex briefly to resuspend the cell pellet in the wash solution. Ensure the cells are fully resuspended. Centrifuge the suspension again at a suitable speed and time (e.g., 1000-1500 rpm for 5-10 minutes) to re-pellet the cells. Repeat the washing.

3. Add ice cold 80% v/v methanol (same volume as the original media) to the cells pellet, vortex the cells for 15 seconds, transfer the methanol-cell suspension to a centrifuge tube (e.g. 2 mL Eppi).  Repeat the extraction with 80% v/v methanol (same volume), vortex, and combine this suspension with the first one in the same centrifuge tube.

4. Store the methanol extract at -20°C or colder until shipment. 

Note: Ammonium bicarbonate solution can be substituted with PBS.

1. Harvest samples. Samples are collected at an OD600 of ca. 0.5, and 2 ml of the culture is vacuum filtered through a filter membrane (e.g. HVLP02500, Merck Millipore).

2. Immerse the filter-containing cells into 1 ml of a pre-chilled solvent mixture (acetonitrile:methanol:H2O in a 40:40:20 ratio) at -20°C.

3. Incubate the filter in the solvent mixture overnight at -20°C for thorough extraction.

4. Centrifuge the cell extracts for 20 minutes at -9°C, at a speed of 13,000 rpm

5. Store extracts at -80°C until shipment

1. Instruct the participant to collect a fresh fecal sample into the sterile container without additives.

2. Emphasize the importance of not contaminating the sample with urine or toilet water during collection.

3. Collect 3 g of the sample.

4. Immediate freezing at -18°C is highly recommended.

5. Transfer samples to -80°C as soon as practically possible and store them until analysis.

Recommended collection kit

1. After removing and washing the tissues with PBS, cut them into smaller pieces and transfer to sample tubes.

2. Immediately snap freeze the tubes in liquid nitrogen.

If immediate freezing is not possible:

1. Cut the tissue into smaller pieces (~1–5 mm³) to enhance solvent penetration.

2. Submerge the tissue in pre-chilled 100% methanol (10–20x the tissue volume).

3. Incubate at -20°C for at least 30 minutes to overnight.

4. Store at -20°C or colder in methanol until further processing.

1. Remove 500 µL of cell culture media from each sample well or plate, transfer to a clean microcentrifuge tube.
   

2. Centrifuge the collected media at 1,000 × g for 5 minutes at 4 °C to pellet cell debris.
   

3. Transfer 200 µL of the clear supernatant to a pre-labeled 2 mL Eppendorf tube (snap-cap or screw-cap recommended).

4. Add 800 µL of methanol to the supernatant (final methanol concentration: 80%).

5. Vortex for 10–15 seconds to mix thoroughly.
   

6. Store the extracted samples at –80 °C until shipment.

   Note: The volumes can be adjusted based on the available media volume, the final methanol concentration should be 80% (v/v)