ensuring that no MCs are aspirated in the process, and

replace with sterile DPBS. Repeat this washing step twice.

(c)

Remove the sample supernatant, as described above, then

add 1 mL of Perm/Stain solution to the tube in one

gentle motion, being sure to resuspend all the MCs in

the process.

(d)

Incubate the sample for 15 min at room temperature.

(e)

Aspirate the supernatant in the tube and use it to resus-

pend the MCs in the tube.

(f)

Incubate for 15 min at room temperature.

(g)

Ensure that the MCs have sedimented, then wash the

permeabilized and stained cell/microcarrier suspension

with DPBS twice as mentioned previously.

(h)

Transfer the permeabilized and stained cell/MC suspen-

sion to the uncoated well of a six-well plate using a 1-mL

pipette and a wide orifice (1.5 mm) pipette tip.

(i)

Add an additional 1 mL of DPBS to each well containing

MC aggregates.

(j)

The permeabilized and stained cell/MC suspension can

now be viewed under the microscope using the correct

fluorescence filter (see Note 1).

(k)

Stained samples can be stored for up to 7 days if wrapped

in aluminum and kept at 4 ^C.

4. Flow cytometry analysis of the sample to determine hMSC

marker expression rates.

(a)

Following the separation of the cells from the MC, dilute

the sample with 1 mL of staining buffer (see Note 22).

(b)

Centrifuge the sample at 400 g for 5 min.

(c)

Remove the supernatant, while ensuring that the cell pel-

let is not aspirated, then resuspend the pellet in 200 μL of

staining buffer (see Note 22). Aliquot the sample and add

the fluorophore-conjugated antibodies listed in Table 2.

(d)

Briefly vortex the samples to ensure homogeneity, then

incubate the samples at 4 ^C for 10 min.

(e)

Dilute each sample by adding 1 mL of staining buffer (see

Note 22) and briefly vortex to interrupt the staining

procedure.

(f)

Centrifuge the samples at 400 g for 5 min.

(g)

Remove the supernatant, while ensuring that the cell pel-

let is not aspirated, then resuspend each pellet in 100 μL of

staining buffer (see Note 22). The samples may now be

analyzed using flow cytometry.

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