NonInvasive Technologies, LLC (03/2007)
SCSR Protocol: Isolation of Colonocytes
from Stool
Materials:
PROCEDURE:
A. Preparation
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)
G. Freeze
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.
H. Clean-up
29. Discard the unused fecal samples,
stomacher bags and filters in biohazard bags.
30.Clean
all areas with bleach.
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Technologies,