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Focus: Cell Proliferation

Compatibility of the CellTiter 96^® AQ_ueous One Solution with the Luciferase Assay System

By Natalie Betz, Ph.D.
Promega Corporation

Introduction

A common goal of many researchers is the analysis of cell proliferation, particularly in cell biology, cancer research and immunology. In addition, the ability to quantitate both cell number and reporter gene expression can be particularly useful to those investigating the effects of cell growth or cell cycle on specific gene expression. The compatibility of CellTiter 96^® AQ_ueous One Solution Assay^(a) (Cat.# G3582) with various Promega luciferase reagents would allow the sequential determination of both cell number and luciferase reporter gene expression on the same transfected cell population. Therefore, experiments were performed to investigate the compatibility of these reagents.

Experiment

Transfection: NIH3T3 cells were plated in a 96 well plate and transfected with the pGL3 Control Vector^(c,d,e) (Cat.# E1741) using TransFast™ Transfection Reagent^(f) (Cat.# E2431) at a DNA:reagent charge ratio of 1:1. pGL-3 Vectors express luciferase. The cells were approximately 70% confluent at the time of transfection. The protocol outlined in the TransFast™ Transfection Reagent Technical Bulletin (#TB260) was followed, and 20% of the volumes recommended for a 24 well plate were used (based on 1µg DNA/well for a 24 well plate). The DNA, medium and TransFast™ Reagent were incubated together for 10 minutes at room temperature. The mixture was added to the cells and incubated for 1 hour at 37°C. The DNA-medium-reagent mixture was removed and complete medium (200µl DMEM containing 10% calf serum) was added to each well. Cultures then were incubated an additional 48 hours at 37°C.

Proliferation/Luciferase Assay: Transfected cells (samples A-F) were re-fed with 100µl (per well) fresh DMEM medium + 10% calf serum. CellTiter 96^® AQ_ueous One Solution was added, 20µl per well, to 12 wells of transfected cells (samples C-F), and to 3 wells containing medium only (sample G). See Table 1 for the experimental design and data. The cells were incubated for 3 hours at 37°C. Absorbance (490nm) was read using a SPECTRAmax^® 250 plate reader.

Table 1. CellTiter 96^® AQ_ueous One Solution Assay and Luciferase Detection in Transfected NIH3T3 Cells.

*Samples were analyzed in triplicate; MTS = CellTiter 96^® AQ_ueous One Solution; absorbance is average (+/-SD); RLU = relative light units; DNA = pGL3-Control Vector.
Note: The CellTiter 96^® AQ_ueous One Solution is bright yellow; it changes phenol red-containing media yellow-brown and changes the same media containing metabolically active cells deep burgundy. 

Medium with or without the CellTiter 96^® AQ_ueous One Solution was removed from row wells A through E and replaced with 125µl of either 1X Reporter Lysis Buffer (RLB; rows A and C) or 1X Cell Culture Lysis Reagent^(e) (CCLR; rows B and E). To the wells in row D, 25µl of 5X Reagent Lysis Buffer (RLB) was added directly to the medium, and to the wells in row F, 25µl of 5X CCLR was added directly to the medium (to 1X final concentration). The plate was incubated for 10 minutes at room temperature with occasional rocking. The solution in each well was mixed by repeated pipetting ("triturated") to aid in cell lysis. Cells in all wells appeared lysed when examined under a microscope; however, for the wells still containing the CellTiter 96^® AQ_ueous One Solution, it was difficult to determine whether lysis had occurred since the dye solution obscured the cells. An aliquot of each sample (20µl or 4µl) was then placed into clean 1.5ml tubes and 100µl of reagent from the Luciferase Assay System^(b) (Cat.# E1500) was added. Light output was read immediately in a Turner TD-20/20 Luminometer (Cat.# E2041) (2-second delay, 10-second read). The 20µl extracts from row wells A and B produced light output >9,999, which is above the linear range of the luminometer. Therefore, 4µl per sample was read instead.

Samples A and B served as the positive controls for the luciferase assay (no CellTiter 96^® AQ_ueous One Solution). Cells lysed in 1X CCLR yielded higher luciferase activity measurements (5,703 RLU) compared to those lysed in 1X RLB (3,674 RLU). This is most likely due to not freeze-thawing the transfected cells, a requirement for many cell types when using RLB as the lysis reagent. When the transfected cells were first treated with the CellTiter 96^® AQ_ueous One Solution, the MTS reagent^(a) was removed and a luciferase assay was performed on the same cells. The RLU values were lower than those compared to no MTS reagent treatment (compare values for C to A and values for E to B). In this experiment, the RLU values were reduced approximately 50%. When the CellTiter 96^® AQ_ueous One Solution was not removed, and 5X Lysis Buffer was added directly to the culture medium (final concentration 1X), the RLU values were reduced even more dramatically, particularly in the case of RLB (compare values for D to C, and values for F to E). It appears that 5X CCLR was more efficient at lysing the transfected cells in the presence of culture medium and the CellTiter 96^® AQ_ueous One Solution, most likely due to the more robust lytic nature of CCLR vs. RLB.

Summary

In general, these results support removing the CellTiter 96^® AQ_ueous One Solution and lysing the transfected cells in 1X RLB or 1X CCLR before performing the luciferase assay. The CellTiter 96^® AQ_ueous One Solution does appear to be compatible with luciferase assay reagents, although a 50% decrease in luciferase activity may be expected when the transfected cells are treated with the MTS reagent prior to measuring luciferase activity. Depending on the level of luciferase reporter gene expression, a 50% reduction in luciferase activity may not be significant and offers the advantage of determining cell number on the same cells in which reporter gene expression was measured.

^(a)The MTS tetrazolium compound is is the subject of U.S. Pat. No. 5,185,450 assigned to the University of South Florida and is licensed exclusively to Promega Corporation.

^(b)U.S. Pat. No. 5,283,179, Australian Pat. No. 649289 and other patents. Certain applications of this product may require licenses from others.

^(c)U.S. Pat. No. 5,670,356.

^(d)The method of recombinant expression of Coleoptera luciferase is covered by U.S. Pat. Nos. 5,583,024,  5,674,713 and 5,700,673.

^{(e)Certain applications of this product may require licenses from others.}

^(f)The cationic lipid component of the TransFast™ Transfection Reagent is covered by U.S. Pat. Nos. 5,824,812,  5,869,715 and pending foreign patents.

CellTiter 96 is a trademark of Promega Corporation and is registered with the U.S. Patent and Trademark Office.

SPECTRAmax is a registered trademark of Molecular Devices Corporation.

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