(h)

For result evaluation using the appropriate software (see

Note 4), first determine the cell population by gating the

events of interest using the Blank sample (see Table 2) and

a forward scatter area (x-axis) versus side scatter area

(y-axis) plot. Then exclude potential cell aggregates from

the cell population by gating the singlet events using a

forward scatter area (x-axis) versus forward scatter height

(y-axis) plot.

(i)

Once the singlet population has been adequately deter-

mined, use the Isotype sample (see Table 2) to gate for

PE⁺, FITC⁺, and APC⁺ events using a PE-, FITC-, or

APC-area (x-axis) versus side scatter area (y-axis) plot.

Gate in such a way that the field contains only 1% of the

stained population. This results in non-specifically stained

cells being excluded from the evaluation process.

(j)

Through application of this gating strategy, the percent-

age of specifically stained cells in the CD⁺ and CD^ sam-

ples (see Table 2) may be determined.

4

Notes

1. Cell attachment and confluency were determined using the

EVOS™FL Auto 2 Imaging System and the corresponding

software DiamondScope v2.0.2094.0 and EVOS Analysis

v1.4.998.659 (Thermo Scientific™Invitrogen™).

2. Cell density, viability, diameter, and aggregation rate were

determined

post-trypsination

using

the

NucleoCounter^®

NC-200™(Chemometec). This system makes use of the fluo-

rescent dyes, acridine orange, and DAPI, to differentiate

between cells with an intact and compromised membrane,

Table 2

Cell suspension and fluorophore-conjugated antibody combinations required for the flow cytometry

analyses

Sample

ID

Cell

suspension

REA control

antibodies

Cluster of differentiation (CD) antibodies

PE

FITC APC

CD105-

PE

CD90-

FITC

CD73-

APC

CD45-

PE

CD36-

APC

Blank

50 μL

Isotype

47 μL

1 μL 1 μL 1 μL

CD⁺

47 μL

1 μL

1 μL

1 μL

CD^

48 μL

1 μL

1 μL

104

Misha Teale et al.