(a)
Transfer the homogenous sample to a 15-mL centrifuge
tube for intermediate storage.
(b)
Gently resuspend the sample using a 10-mL sterile pipette
and aliquot the sample into 2 2 mL and 1 1.5 mL
microtubes, as shown in Fig. 4.
(c)
Allow the MCs to sediment in all containers.
(d)
Transfer the supernatant from one 2-mL microtube to a
new 1.5-mL microtube in such a way as to not aspirate any
MCs. This supernatant may then be analyzed to determine
substrate and metabolite concentrations (see Note 3).
(e)
To the 1.5-mL microtube, add 1 mL of formalin. This
sample, henceforth referred to as aliquot 1, may then be
used to determine cell distribution, as well as MC aggre-
gation (see Note 19).
(f)
Using 1 mL of pre-warmed DPBS for each, resuspend the
sedimented MCs in both 2-mL microtubes.
(g)
Allow the MCs to sediment, then remove and replace the
supernatant with 0.5 mL of pre-warmed TrypLE Select
1.
(h)
Place the 2-mL microtubes horizontally on an orbital
shaking platform at 130 rpm, 25 mm, 37 C for 7 min
(see Note 7).
(i)
Following incubation, resuspend the suspension in both
2-mL microtubes 10 times using a 1-mL pipette and
standard pipette tip, then use aliquot 3 to determine cell
density (see Note 2). Be sure to account for the concen-
tration factor (approximately 10:1, see Note 13).
(j)
Based on the cell density results above, use a 70-μm cell
strainer to filter the volume of aliquot 2 corresponding to
a maximum of 1.5 106 cells into a 1.5-mL microtube
and separate them from the MCs (aliquot 3 may also be
used in combination with aliquot 2, if the latter’s volume
proves insufficient). Then proceed with the flow cytome-
try analysis (see Note 4).
3. Determining cell distribution and MC aggregation by staining
with DAPI.
(a)
Prepare the Perm/Stain working solution by adding
DAPI working solution (see Note 20) to the Triton®
X-100 working solution (see Note 21) at a ratio of
2.5:1000. In other words, 2.5 μL of DAPI working solu-
tion for every 1,000 μL of Triton® X-100 working
solution.
(b)
Following fixation with formalin of the sample in the 1.5-
mL Eppendorf tube, remove the supernatant, while
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