Cytotoxicity Assays with Zebrafish Cell Lines

Summary

This protocol presents commonly used cytotoxicity assays (Alamar Blue [AB], CFDA-AM, Neutral Red, and MTT assays) adapted for the assessment of cytotoxicity in zebrafish embryo (ZEM2S) and liver (ZFL) cell lines in 96-well plates.

Abstract

Fish cell lines have become increasingly used in ecotoxicity studies, and cytotoxicity assays have been proposed as methods to predict fish acute toxicity. Thus, this protocol presents cytotoxicity assays modified to evaluate cell viability in zebrafish (Danio rerio) embryo (ZEM2S) and liver (ZFL) cell lines in 96-well plates. The cytotoxicity endpoints evaluated are mitochondrial integrity (Alamar Blue [AB] and MTT assays), membrane integrity via esterase activity (CFDA-AM assay), and lysosomal membrane integrity (Neutral Red [NR] assay). After the exposure of the test substances in a 96-well plate, the cytotoxicity assays are performed; here, AB and CFDA-AM are carried out simultaneously, followed by NR on the same plate, while the MTT assay is performed on a separate plate. The readouts for these assays are taken by fluorescence for AB and CFDA-AM, and absorbance for MTT and NR. The cytotoxicity assays performed with these fish cell lines can be used to study the acute toxicity of chemical substances on fish.

Introduction

Chemical substances need to be tested regarding their safety for human health and the environment. Molecular and cellular biomarkers have been increasingly considered in safety assessments to predict effects on living organisms by regulatory agencies and/or legislations (e.g., REACH, OECD, US EPA)^1,2, since they can precede the in vivo adverse outcome (e.g., endocrine disruption, immunological response, acute toxicity, phototoxicity)^3,4,5,6,7

Protocol

NOTE: See the Table of Materials for the list of materials used in this protocol and Table 1 for the composition of solutions and media used in this protocol.

1. Preparing ZFL and ZEM2S cells

1. Start with a T75 flask of ZFL or ZEM2S cells with 80% confluence, cultured in the respective complete medium at 28 °C without CO₂.
2. Remove the culture medium from the flask and wash the cells by adding 10 mL of 1x phosphate-buffered saline (PBS) (0.01 M). Add 3 mL of 1x trypsin (0.05% v/v; 0.5 mM trypsin-EDTA) to the culture flasks. Incubate at 28 °C for 3 min.

Results

Figure 3 shows the plates of the AB, CFDA-AM, NR, and MTT assays. For the AB assay (Figure 3A), the blank wells and wells with no or a reduced number of viable cells show blue color and low fluorescence, while the wells with a high number of viable cells are pinkish and present high fluorescence values due to the transformation of resazurin (AB) into resorufin (pinkish substance) by the viable cells. For the CFDA-AM assay, there is no visible difference in the c.......

Discussion

Cytotoxicity assays are widely used for in vitro toxicity evaluation, and this protocol article presents four commonly used cytotoxicity assays modified to be performed in zebrafish cell lines (i.e., cell density for 96-well plate, incubation time in the MTT assay, FBS depletion during the chemical exposure condition, and maximal acceptable concentration for the SC). As these assays quantify cytotoxicity by different cell viability endpoints (metabolic function, lysosomal membrane integrity, and cell membrane in.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
5-CFDA, AM (5-Carboxyfluorescein Diacetate, Acetoxymethyl Ester)	Invitrogen	C1345
Cell culture plate, 96 well plate	Sarstedt	83.3924	Surface: Standard, flat base
DMEM	Gibco	12800-017	Powder, high glucose, pyruvate
FBS - Fetal Bovine Serum, qualified, USDA-approved regions	Gibco	12657-029
Ham's F-12 Nutrient Mix, powder	Gibco	21700026	Powder
HEPES (1 M)	Gibco	15630080
Leibovitz's L-15 Medium	Gibco	41300021	Powder
Neutral red	Sigma-Aldrich	N4638	Powder, BioReagent, suitable for cell culture
Orbital shaker	Warmnest	KLD-350-BI	22 mm rotation diameter
Dulbeccos PBS (10X) with calcium and magnesium	Invitrogen	14080055
Penicillin-Streptomycin (10,000 U/mL)	Gibco	15140122
Resazurin sodium salt	Sigma-Aldrich	R7017	Powder, BioReagent, suitable for cell culture
RPMI 1640 Medium	Gibco	31800-014	Powder
Sodium bicarbonate	Sigma-Aldrich	S5761	Powder,  bioreagent for molecular biology
Thiazolyl Blue Tetrazolium Bromide  98%	Sigma-Aldrich	M2128
Trypan blue stain (0.4%)	Gibco	15250-061
Trypsin-EDTA (0.5%), no phenol red	Gibco	15400054
ZEM2S cell line	ATCC	CRL-2147	This cell line was kindly donated by Professor Dr. Michael J. Carvan (University of Wisconsin, Milwaukee, USA)
ZFL cell line	BCRJ	256