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Preparation of Cells Proliferation Curve Cultured in NCP
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Introduction

Spheroid proliferation assay using tetrazolium or resazurin might have a little problem. Because it is difficult that these molecules penetrate into center of spheroid, reads to inaccurate detection results. Therefore, we recommend Luminescent ATP assay as a simple, reproducible and accurate method for quantification of spheroid viability. Here, we introduce a protocol for evaluation of cell viability and preparation of cells proliferation curve cultured in NanoCulture Plate.

Materials, reagents and equipment

1. Cell
HepG2(HepG2 human hepatocellular carcinoma cell) ATCC Cat# HB-8065™
2. Reagents
Media EMEM supplemented 10%FBS and antibiotics
(Heat-inactivated FBS must be used)
CellTiter-Glo ®Luminescent Cell Viability Assay Promega Cat# G7570
3. Materials
NanoCulture Plate MS pattern,
Low-Binding, 96-well
ORGANOGENIX Cat# NCP-LS96
(Qty: 4, Individual plates are necessary every measurement day.)
4. Apparatus
Luminometer TECAN infinite M200 or any similar kind

Methods

【3D Cell Culture】

1. Add media (50 µL/well) in NCP
2. Centrifuge for 5 minutes at 1,000xg to remove bubbles from the edge of the well bottom.
3. Stand for 15 – 20 minutes, at room temperature or inside the CO2 incubator to remove micro-bubbles
4. Prepare HepG2 cell suspension (1×104 cells/ 50 µL)
5. Add HepG2 cell suspension (1×10 4cells/50 µL/well)in NCP(total volume of media will be 100 L/well).Please try not to scratch well bottom which may disturb cell spheroid growth. It may results monolayer cell growing.
6. Stand for 20 – 30 minutes at ambient condition after seeding cells for better cell attachment on the plate. If NCP plate place inside the CO2 incubator before the cells attach to NCP, it may result in an uneven distribution of spheroids due to vibration and convection micro-currents while moving the plates.
7. We strongly recommend to pour autoclaved water(about 100µL) between the well to minimize the edge effect.
8. Incubate at 5%CO2, 37℃
9. Observe cell morphologies and measure cell proliferation by ATP assay according to experimental purpose. (For proliferation curve, we recommend to measure ATP concentration on day 1, 3, 5 and 7)

【Preparation of CellTiter-Glo (CTG) reagent】

10. Mix CTG Buffer and CTG Substrate, and stock at -20℃ in small quantity
11. Thaw at time of use

【Preparation of ATP Samples for standard curve】

12. Prepare ATP standard solution with distilled water
13. Add media (90 µL) and CTG reagent (100 µL) to blank well in NCP (same plate with the real samples)
14. Add ATP standard solution (10 µL) to 13

【ATP assay】

15. Add CTG reagent (100 µL) to samples in NCP
16. Vortex-mix for 10 minutes and stand at 37℃ for 20 minutes
17. Measure luminescence
18. Caculate ATP amount of each well contains spheroid sample from the standard curve

Example of Results

Cell Proliferation Curve of HepG2 and Microscopic Images