8. To stain cells, let microcarriers settle for 2 min, aspirate super-

natant without disturbing the microcarriers and rinse once with

PBS. Aspirate PBS and add 100 μL Calcein AM staining solu-

tion. Incubate at room temperature for 30 min, observe and

image cells under fluorescent microscope with a blue filter

(Fig. 5).

9. To enumerate cells, centrifuge cell-microcarrier sample at

179
g, 2 min, discard supernatant. Add 10 mL working 3D

FloTrix^® Digest solution and incubate in 37 ^C water bath.

Mix thoroughly by pipetting for 30 s at every 10-min interval

to facilitate dissolution of microcarriers (Fig. 6). At 30 min (see

Note 12), centrifuge at 179
g, 5 min, discard supernatant

and resuspend in 10 mL PBS.

Fig. 5 Calcein-AM staining of live cells on microcarriers at 24 h, 48 h, and 72 h

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Huanye Xu et al.