Viability Assays for Cells in Culture

Summary

Therapeutic compounds are often first examined in vitro with viability assays. Blind cell counts by a human observer can be highly sensitive to small changes in cell number but do not assess function. Computerized viability assays, as described here, can assess both structure and function in an objective manner.

Abstract

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized viability assays are expensive in terms of equipment but can be faster and more objective than manual cell counts. The present report describes the use of three such viability assays. Two of these assays are infrared and one is luminescent. Both infrared assays rely on a 16 bit Odyssey Imager. One infrared assay uses the DRAQ5 stain for nuclei combined with the Sapphire stain for cytosol and is visualized in the 700 nm channel. The other infrared assay, an In-Cell Western, uses antibodies against cytoskeletal proteins (α-tubulin or microtubule associated protein 2) and labels them in the 800 nm channel. The third viability assay is a commonly used luminescent assay for ATP, but we use a quarter of the recommended volume to save on cost. These measurements are all linear and correlate with the number of cells plated, but vary in sensitivity. All three assays circumvent time-consuming microscopy and sample the entire well, thereby reducing sampling error. Finally, all of the assays can easily be completed within one day of the end of the experiment, allowing greater numbers of experiments to be performed within short timeframes. However, they all rely on the assumption that cell numbers remain in proportion to signal strength after treatments, an assumption that is sometimes not met, especially for cellular ATP. Furthermore, if cells increase or decrease in size after treatment, this might affect signal strength without affecting cell number. We conclude that all viability assays, including manual counts, suffer from a number of caveats, but that computerized viability assays are well worth the initial investment. Using all three assays together yields a comprehensive view of cellular structure and function.

Introduction

The most common viability assay in the biological sciences involves cell counts. This is evidenced by an analysis of the top (most recent) 200 publications that appeared in PubMed with either of the keywords “in vitro” or “culture” on 4/29/2013 and 4/30/2013. Of these publications, 23.5% used cell count assays, including manual cell number counts, automated cell number counts with imaging software, and Trypan blue exclusion. The Live/Dead assay was used in 1% of these publications. The number of publications using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay for metabolic viability was 11%. This survey o....

Protocol

A schematic of the protocols is illustrated in Figure 1.

1. Cell Plating

Plate cells in 96-well plates at different plating densities (Figure 2). For linearity checks on the N2a neuroblastoma cell line, plate 2.5k, 5k, 10k, and 15k cells per well in 3 or 6 wells/group. For linearity checks in rat primary cortical neurons, plate 25k, 50k, 100k, and 200k cells per well in 3 or 6 wells/group. If the cell lines or primary cells of interest look healthy at different plating densities, plate at and around the optimal cell density for that cell type.

Note: In t....

Results

The rate-limiting factor in these experiments is the infrared staining, as the ATP assay is relatively brief in duration. For the infrared assays, we anticipate that eight 96-well plates can be stained and scanned within one day by staggering two batches of four plates each (see Figure 1). This estimation assumes 20 min of fixation, 30 min of washing, 30 min of blocking, 2 hr primary antibody incubation followed by 30 min of washes, 1 hr secondary antibody incubation followed by 30 min of washes, 30 min .......

Discussion

We have found that signal strength in all three viability assays is linear and correlated with plating density. However, not all the assays are equally sensitive to 2-fold or 1.5-fold changes in plating density. For N2a cells, the infrared assays are less sensitive than the ATP assay, particularly at lower plating densities. Although the infrared assays are less sensitive than ATP, the DRAQ5 + Sapphire assays and the α-tubulin assays are in good agreement in that they reveal the highly protective impact of N-acetyl .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Cell Titer Glo	Promega	G7572	Buy in 100 ml quantities and aliquot, instead of purchasing the more expensive 10 ml quantity. Reconstituted, unused reagents can be refrozen at -20 °C for at least 21 weeks
18% Formalin	Thermo-Shandon	9990244	Buying this fixative avoids the weighing out of formaldehyde powders and boiling of the solution; exposure to vapors is thereby minimized
Sucrose	Sigma-Aldrich	S0389	It is not essential to add this to formaldehyde solutions but it improves the appearance of the fixed cells
Odyssey Block	LI-COR	927-40003	This fish serum can be bought in bulk and frozen at -20 °C for long term use
Triton-X 100	Sigma-Aldrich	21568	We store a stock solution of 10% Triton-X 100 in sterile water at 4 °C
Sodium Phosphate Monobasic	Fisher	S468	One can also buy PBS tablets or 10x PBS solutions, but they are more expensive
Sodium Phosphate Dibasic	Fisher	S373	See above
Sodium Azide (250x)	Ricca Chemical Company	7144.8-16	Do not buy the powder because sodium azide is very toxic. We store all our used antibodies in 1x sodium azide at 4 °C until they become contaminated with debris
Mouse anti-α-tubulin	Sigma-Aldrich	T5168	This antibody is expensive but can be greatly diluted and is highly specific
Mouse anti-MAP2	Sigma-Aldrich	M9942	This antibody is expensive but is highly specific (a prerequisite for In-Cell Westerns)
800 nm Goat anti-mouse IgG	LI-COR	926-32210	Other companies also sell infrared secondary antibodies. Be sure to purchase the highly cross-adsorbed antibodies and note that concentrations of IgGs may vary with the source
DRAQ5	Biostatus	DR50200	This compound used to be sold by LI-COR at 1 mM
Sapphire	LI-COR	928-40022
Luminometer	PerkinElmer	VICTOR3 1420 multilabel counter
Odyssey Imager	LI-COR	9201-01
Shaker/Mixer	Research Products International	248555