NonInvasive Technologies, LLC (03/2007)
SCSR Protocol: Isolation of Colonocytes from Stool
1. Store the stool samples collected in 15 ml of transport medium in pre-weighed 30 ml screw capped fecal collection tubes. (We recommend the samples be processed within 7 days of collection. If the samples cannot be processed immediately, store them at 4șC. Samples can be transported at room temperature a maximum of 7 days. The 15 ml volume is designed to preserve a 0.5 - 1.0 g sample. )
2. Prepare yourself by putting on gown, gloves and mask.
3. Pre-warm the SCSR dispersing medium (SCSR-T) and cushion solution (SCSR-C) to room temperature.
4. Record the weight of the tubes containing the fecal samples.
5. Calculate the weight of the fecal sample by subtracting the weight before collection from the weight after collection (If the total weight of the fecal sample is more than 1 g, add 10 ml of transport medium (SCSR-T) to the tube containing sample. This will speed up filtration in Step 10. )
B. Strain and Filter Sample
6. Make sure the sample tubes are tightly closed and do not leak, then vortex to suspend the sample.
7. Working in the hood, transfer the sample into a 330 ”m Stomacher strainer bag (see photo Section 3.1).
8. Press the bag to help the sample through the strainer into the outer bag (see photo, Section 3.1).
9. Draw out filtered sample using a plastic 25 ml pipette from the plastic bag into a 50 ml labeled tube (see photo, Sect 3.1).
10. Filter the sample through 40 ”m filter into pre-labeled 50 ml centrifuge tube. If this process is slow, use pipette to unclog the filter by repeatedly aspirating and dispensing the sample into the filter. It may be helpful to tip the filter at a 30deg angle (see photo, Section 3.2).
11. Calculate the volume of the sample equivalent to 0.5 g of the original fecal sample.
12. Remove the filtered sample equivalent to 0.5 g of fecal sample into another 50 ml tube using a pipette.
C. Underlay Cushion and Centrifuge
13. Adjust the total volume to 25 ml with transport medium (SCSR-T) and mix by vortexing.
14. Carefully underlay the sample with 10 ml cushion solution (SCSR-C), pre-warmed to room temperature using a 10 ml pipette (see photo, Section 3.3).
15. Spin the samples at 200xg for 10 minutes at room temperature (see photo, Section 3.4).
D. Extract Fractions
16. Remove supernatant (if desired, save an aliquot for studying extracellular microbes), and discard into bleach-containing waste bottle. (To maximize cell recovery, leave approximately 5 ml of supernatant above the interphase.) (see photo, Section 3.5)
17. Carefully remove the interphase into a pre-labeled sterile 50 ml tube. (To maximize cell recovery, it is permissible to include 5 ml of the supernatant from above the interphase. It is permissible to include a small amount of the cushion with the interphase.) (see photo, Section 3.6)
18. Leave the cushion in the tube containing the pellet. The cushion will be washed away in subsequent steps.
E. Wash Cell Fractions
19. Wash cell fractions (ie interphase and pellet fractions) by adding cold PBS pH 7.2 to each of the tubes, adjusting the total volume to 40 ml. Re-suspend by either inverting the tubes several times or gently vortexing. Spin the cells at 900xg at 4șC for 10min. Carefully aspirate the supernatant and dispense into a container containing bleach.
20. Repeat wash twice (Step 19), using 15 ml aliquots of PBS.
21. Re-suspend the pellet in 1 ml cold PBS per 0.5 g of original fecal sample and place the cell suspension on ice.
F. Count Cells and Aliquot
22. To disaggregate clumped cells, pass cell suspension through a 20 G needle 10 times while the tubes are on ice.
23. Mix the cells by tapping the tube gently to make the cells suspension homogenous.
24. Count the cells in a Coulter counter using 2-5 ”m and 5-8 ”m ranges.
25. Prepare aliquots according to desired cell counts. (We recommend using the 5-8 ”m cell counts as a basis)
26. Spin the tubes containing cells in a refrigerated centrifuge at 900xg for 5 min.
27. Remove supernatant and re-suspend cells in 100 ”l serum-free cell freezing medium. Make sure to suspend the pellet by gently mixing after adding the freezing medium.
28. Store the aliquots of colonocytes at -80șC.
29. Discard the unused fecal samples, stomacher bags and filters in biohazard bags.
30.Clean all areas with bleach.