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Protocol

Streamlined High Throughput Drug Screening with PrimeSurface® White 384 well 3D plates

R&D Team, PHCNA

Aug 27, 2021

Abstract

Using PrimeSurface® white plate, cancer spheroid cells can be formed and tested for drug screening using luminescence viability assay in the same well without the need for reagent transfer. This streamlined process shortens the experimental steps and time required in drug testing thus providing a robust, reproducible and reliable assay. In this technical note, we demonstrate cell viability of 4 different tumor cells and the streamlined method of intracellular ATP measurement using CellTiter Glo® (Promega Co., Ltd).


Introduction

Using PrimeSurface® white plate, cancer spheroid cells can be formed and tested for drug screening using luminescence viability assay in the same well without the need for reagent transfer. 


Reagents and Equipment

  1. RPMI1640 (+10%FBS +1%Penicillin-Streptomycin Mixed Solution) 
  2. CellTiter Glo® Luminescent Cell Viability Assay Cat. No. G7572 (Promega Co., Ltd.) 
  3. PrimeSurface® 384 well clear plate (MS-9384UZ, Sumitomo Bakelite Co., Ltd.) 
  4. PrimeSurface® 384 well white plate (MS-9384WZ, Sumitomo Bakelite Co., Ltd.) 
  5. Plate Reader: Fusion α-FP (Perkin Elmer Co., Ltd ) 

Procedure

 

Different types of Cancer cell lines can form tight, compact or loose spheroids Four types of cells, shown below serve as a representative cases for spheroid formation. Using CellTiter Glo®, a homogeneous "add-mix-measure" reagent results in cell lysis and generation of a luminescent signal proportional to the amount of ATP present which is directly proportional to the number of cells present in culture. 

Cell Proliferation Curve 

Cells were seeded in PrimeSurface® 384 well white plate (Cat. No. MS-9384WZ) with a density of 250, 500 and 1000 cells/well in 25μL of culture medium*1. Cells were incubated at 37℃ / 5% CO2 . Luminescent intensities were measured every two days after addition of 25μL volume of CellTiter Glo® reagent and ten minutes standing at R.T.*2 

*1: Based on the type of cells, number of cells, and culture medium, the amount of culture medium may need to be adjusted. 

*2: If cell solubility is poor, dissolve the cells using a shaker.

Cell proliferation curve of HepG2, HeLa, MDA-MB-231 and ACHN cells (◆:250cells/well, ■:500cells/well, ▲:1000cells/well) The amount of ATP increased in the case of HepG2, HeLa, and ACHN cells with all cell seeding densities over the 7-days culture period. On the other hand, MDA-MB-231 did not show much increase in the ATP amount. 

Calculation of Z’-Factor

  1. Cells were seeded with 25μL/well media in PrimeSurface® 384 well white plate (MS-9384WZ) as stated above. First column and last column served as the control wells with no cells and culture medium only.
  2.  The intracellular ATP amount was measured at Day5. 25 μL of CellTiter Glo® reagent was added to each wells. 
  3. Z’-factor values were calculated as 100% and 0% and respectively, in wells with and without cells. 

Time Taken

Variable

References

Not available


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