- 3D experiment in 96-well ultra-low adhesion plate from Corning
- Cell line: PANC-1 5000 cells/well; P39
- Viability: 74 %, 86 %, 82 %
- Nanoparticles: GGAG:Ce@SiO2-RB
- Concentration: 0.01 mg/mL = 10 µg/mL
- Growing spheroids 48 hours
- Incubation nanoparticles plan: 48h, 24h, 6h, 2h, 30min, 0h
- Incubation nanoparticles before imaging: 49,5h; 26,5h; 10,5h; 5h;
3h; 0h
- 5 day experiment
Experiment procedure
Day 1, 2, 3 - Cell seeding
- 96-well ultra-low adhesion plate
- seeding cells 5000 cells/well in 100 μL culture medium
- 48h incubation: growing from 14:25 14.11. for 48h
- 24h incubation: growing from 14:50 15.11. for 48h
- other incubation: growing from 8:50 16.11. for 48h or more
Day 3 - Adding NPs for
48h incubation
- solution for pipetting NPs 2x 10 μg/ml = 20 μg/ml
- NPs stock solution in PBS 1 mg/ml; 15 min sonification
- NPs solution volume 1 ml; 20 μg 1mg/ml + 980 μL medium; shaking
before using
- add 100 μL NPs solution to each well containing 100 μL of medium
from growing
- add 100 μL medium to 48h CTRL wells containing 100 μL of medium from
growing
- mix gently the medium in wells - pipette up and down
- growing from 14:25 14.11. for 48h
- incubation started at 15:35 16.11. for 48h
Day 4 - Adding NPs for
24h incubation
- solution for pipetting NPs 2x 10 μg/ml = 20 μg/ml
- NPs stock solution in PBS 2 mg/ml; 15 min sonification
- NPs solution volume 2 ml; 20 μg 2mg/ml + 1980 μL medium; shaking
before using
- add 100 μL NPs solution to each well containing 100 μL of medium
from growing
- add 100 μL medium to 24h CTRL wells containing 100 μL of medium from
growing
- mix gently the medium in wells - round circle with pipette tip
- growing from 14:50 15.11. for 48h
- incubation started at 15:20 17.11. for 24h
Day 5 - Adding
NPs for 6h, 2h, 30min incubation
- solution for pipetting NPs 2x 10 μg/ml = 20 μg/ml
- stock solution and NPs solution prepared for each incubation time
separately
- NPs stock solution in PBS 1 mg/ml; 15 min sonification
- NPs solution volume 1 ml; 20 μg 1mg/ml + 980 μL medium; shaking
before using
- add 100 μL NPs solution to each well containing 100 μL of medium
from growing
- add 100 μL medium to CTRL wells containing 100 μL of medium from
growing
- mix gently the medium in wells - round circle with pipette tip
- growing from 8:50 16.11. for 48h or more
- incubation started at 9:25 18.11. for 6h
- incubation started at 13:55 18.11. for 2h
- incubation started at 15:05 18.11. for 30min
Day 5 - Imaging with
confocal microscope
Real incubation time with
NPs
- Imaging at 17:00 48h (incubation started at 15:35) -> 49,5h
- Imaging at 17:40 24h (incubation started at 15:20) -> 26,5h
- Imaging at 20:00 6h (incubation started at 9:25) -> 10,5h
- Imaging at 19:20 2h (incubation started at 13:55) -> 5h
- Imaging at 18:00 30min (incubation started at 15:05) -> 3h
Data
