20200816 - TH analysis of BEK pilot nanogel plate

Experiment shows successful uptake of the nanogel (cytoplasmic fluorescence intensity).

14 500 cells/well seeded
500 cells/well seeded 1000 cells/well seeded 2000 cells/well seeded 3000 cells/well seeded
2.5 2.5 2.5 2.5
Control Control Control Control
1000 cells/well seeded
1 1 1 1
10 10 10 10 13 2000 cells/well seeded
2 100 2 100 2 100 2 100

ln(nanogel intensity)
1000 1000 1000 1000 3000 cells/well seeded
density

1.5 1.5 1.5 1.5

12

1 1 1 1

11
0.5 0.5 0.5 0.5

0 0 0 0
9 10 11 12 13 14 15 16 9 10 11 12 13 14 15 16 9 10 11 12 13 14 15 16 9 10 11 12 13 14 15 16 10
control control 1 10 100 1000
(wash) (no wash)
ln(nanogel intensity)
nanogel dose (units?)

Nanogel uptake has no significant effect on CDK2 activity in this experiment.

500 cells/well seeded
1.8
1000 cells/well seeded
500 cells/well seeded 1000 cells/well seeded 2000 cells/well seeded 3000 cells/well seeded
4 3.5 3 2.5
1.5 2000 cells/well seeded
Control Control Control Control
1 1 1 1
3.5
10
3 10 2.5 10 10 3000 cells/well seeded

DHB cyto/nuc ratio

100 100 100 2 100 1.2
3 1000 1000 1000 1000
2.5
2
density

2.5
2
1.5 0.9
2 1.5
1.5
1.5
1 0.6
1
1
1
0.5 0.3
0.5
0.5 0.5

0 0 0 0
0.0
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 control control 1 10 100 1000
(wash) (no wash)

DHB cyto/nuc ratio nanogel dose (units?)

Gated histograms
3

2.5
Nanogel-
Nanogel+ Further gating shows that cells taking up nanogel have no distinguisable
2
difference in CDK2 activity from cells that did not take up nanogel. This
sensor is challenging to analyze by fixed-cell experiments, as it’s a dynamic
density

1.5

1
readout of cell-cycle progression. Thus, we don’t know if these cells are on
0.5
their way to exiting the cell cycle or if they will continue building CDK2 activity
0
after prolonged intracellular exposure to the nanogels.
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8

DHB cyto/nuc ratio

Representative example: well B07
(2000 cells/well; 10 units nanogel)
Used this as an example because this dose
yields enough cells in both the nanogel(-) and
nanogel(+) populations after gating. Higher doses
result in almost all cells taking up the nanogel,
leaving nothing to compare them to.