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Adaptation of Stem Cells to 96‐Well Plate Assays: Use of Human Embryonic and Mouse Neural Stem Cells in the MTT Assay

互联网2013-12-31

1957

Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

Human embryonic stem cells (hESC) are difficult to adapt to 96?well plate assays, such as the MTT assay, because they survive best when plated as colonies, which are not easily counted and plated accurately. Two methods were developed to address this problem. In the first, ROCK inhibitor (ROCKi) was used, which allows accurate counting and plating of single hESC. In the second, small colonies were plated without ROCKi but with adaptations for accurate counting and plating. The MTT assay was also adapted for use with mouse neural stem cells. These methods allow the MTT assay to be conducted rapidly and accurately with high reproducibility between replicate experiments. When screening volatile chemicals in a 96?well plate, vapor effects may occur and dose ranges must be carefully defined. The methods were validated using the NIH assay guidance tool. These methodss could readily be translated to other 96?well plate assay. Curr. Protoc. Stem Cell Biol. 23:1C.13.1?1C.13.21. © 2012 by John Wiley & Sons, Inc.

Keywords: human embryonic stem cells; mouse neural stem cells; MTT assay; 96?well plate assays; toxicology

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Introduction
Basic Protocol 1: Preparing mNSC for the MTT Assay
Support Protocol 1: Determining Cell Concentration with a Hemacytometer or a Spectrophotometer
Support Protocol 2: Standard Curve Generation for Counting Cells with a Spectrophotometer
Support Protocol 3: Plating mNSC for the MTT Assay
Basic Protocol 2: MTT Assay
Support Protocol 4: Assay Validation for the MTT Assay with mNSC: Plate Uniformity and Signal Variability
Basic Protocol 3: Preparation of hESC for the MTT Assay Using the Single‐Cell Method with Rock Inhibitor
Alternate Protocol 1: Preparation of Human Embryonic Stem Cells for the MTT Assay Using the Small‐Colony Method
Support Protocol 5: Assay Validation for the Small‐Colony Procedure with hESC: Plate Uniformity and Signal Variability
Reagents and Solutions
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol 1: Preparing mNSC for the MTT Assay

Materials

mNSC (unit 2.6 ) growing in 25‐cm² tissue culture flasks
Dulbecco's phosphate buffered saline, pH 7.4 (DPBS; Invitrogen, cat. no. 25200‐056): pH 7.4
0.05% trypsin‐EDTA (Invitrogen, cat. no. 25200‐056) prepared in DPBS
mNSC culture medium (see recipe )

15‐ml conical centrifuge tubes (BD Biosciences, cat. no. 352097)
Centrifuge
25‐cm² (T‐25) tissue culture flasks (Thermo Scientific, cat. no. 136196z)

Support Protocol 1: Determining Cell Concentration with a Hemacytometer or a Spectrophotometer

Materials

75% ethanol
mNSC culture medium (see recipe )
0.4% trypan blue (optional; Invitrogen, cat. no. 15250)

Hemacytometer
Microscope

Additional reagents and equipment for preparing cells for MTT assay with mNSC ( protocol 1 )

Support Protocol 2: Standard Curve Generation for Counting Cells with a Spectrophotometer

Materials

mNSC culture (80% to 85% confluent; see protocol 1 )
Phosphate‐buffered saline (PBS; e.g., Invitrogen), pH 7.4
mNSC culture medium (see recipe )
Spectrophotometer (e.g., Thermo Scientific BioMate) and cuvettes

Additional reagents and equipment for preparing and trypsinizing mNSC ( protocol 1 ) and counting cells using a hemacytometer ( protocol 2 )

Support Protocol 3: Plating mNSC for the MTT Assay

Materials

mNSC culture medium (see recipe )
mNSC culture (80% to 85% confluent; see protocol 1 )
Phosphate‐buffered saline (PBS; e.g., Invitrogen), pH 7.4
96‐well plates (BD Biosciences, cat. no. 353936)

Additional reagents and equipment for preparing and trypsinizing mNSC ( protocol 1 )

Basic Protocol 2: MTT Assay

Materials

Test chemicals (e.g., phenol; Sigma, cat. no. W322318)
mNSC culture medium (see recipe )
mNSC ( protocol 4 ) or hESC ( protocol 7 or protocol 8 ) growing in 96‐well plates
MTT (Sigma, cat. no. M5655‐1G; stored in the dark; keep room lights off while working with MTT)
Dimethylsulfoxide (DMSO; Fisher, cat. no. D128‐500)

0.22‐µm syringe filters (Pall Life Sciences, cat. no. pn4612)
Syringes (BD, cat. no. 309604)
Microsoft Excel or equivalent spreadsheet software
GraphPad Prism or equivalent data analysis software

Support Protocol 4: Assay Validation for the MTT Assay with mNSC: Plate Uniformity and Signal Variability

Materials

mNSC (unit 2.6 )
mNSC culture medium (see recipe )
Test chemicals (e.g., phenol; Sigma, cat. no. W322318)
MTT (Sigma, cat. no. M5655‐1G) dissolved in PBS at 5 mg/ml. Should be stored in dark and room lights should be off, while working with MTT.
DMSO (Fisher, cat. no. D128‐500)

96‐well plates (BD Biosciences, cat. no. 353936)
NIH Assay Guidance Tool: download from http://assay.nih.gov/assay/index.php/Assay_Validation_2011 (under Navigation menu)

Additional reagents and equipment for plating mNSC ( protocol 4 ) and MTT assay ( protocol 5 )

Basic Protocol 3: Preparation of hESC for the MTT Assay Using the Single‐Cell Method with Rock Inhibitor

Materials

Matrigel (BD Biosciences, cat. no. 354234); store frozen—Matrigel should be diluted 1:2 in DMEM/F12, aliquotted, and frozen; work quickly to ensure the Matrigel does not thicken
DMEM/F12 medium (e.g., Invitrogen)
hESC (H9 line from WiCell; also see Lin and Talbot, , and unit 1.5 in this manual)
mTeSR culture medium kit (includes basal medium plus supplements; Stem Cell Technologies, cat. no. 05850)
10 mM ROCK inhibitor (ROCKi, Y‐27632; Tocris Bioscience, cat. no. 1254) stock solution
Phosphate‐buffered saline (PBS; e.g., Invitrogen), pH 7.4
DMEM medium (serum‐free; e.g., Invitrogen)
4% (w/v) trypan blue
Accutase (eBioscience, cat. no. 00‐4555‐56)
Glass beads (3 mm diameter, e.g., Fisher Scientific, cat. no. 11‐312), sterile
Test chemical (e.g., phenol, Sigma Aldrich, cat. no. W322318)
Dimethyl sulfoxide (DMSO) (Fisher Scientific, cat. no. D128‐500)

Tissue culture treated 6‐well and 96‐well flat bottom plates (BD Falcon, cat. nos. 353046 and 353072)
15‐ml conical centrifuge tubes (BD Biosciences, cat. no. 352097)
Centrifuge
3‐ml syringes (BD Biosciences, cat. no. 309604)
Acrodisc syringe filters (Pall, 0.2 µm, 25 mm, cat. no. PN 4612)

Additional reagents and equipment for culture of hESC (Lin and Talbot, ; unit 1.5 ), counting cells using a hemacytometer ( protocol 2 ), and MTT assay ( protocol 5 )

Alternate Protocol 1: Preparation of Human Embryonic Stem Cells for the MTT Assay Using the Small‐Colony Method

Materials

mTeSR culture medium kit (includes basal medium plus supplements; Stem Cell Technologies, cat. no. 05850)
Accutase (eBioscience, cat. no. 00‐4555‐56)
Glass beads (3 mm diameter, e.g., Fisher Scientific, cat. no. 11‐312), sterile
Phosphate‐buffered saline (PBS; e.g., Invitrogen), pH 7.4
Test chemicals
Dimethyl sulfoxide (DMSO)

Tissue culture–treated 6‐well and 96‐well flat‐bottom plates (BD Falcon, cat. nos. 353046 and 353072)
15‐ml conical centrifuge tubes (BD Biosciences, cat. no. 352097)
Inverted phase‐contrast microscope
3‐ml syringes (BD Biosciences, cat. no. 309604)
Acrodisc syringe filters (Pall, 0.2 µm, 25 mm, cat. no. PN 4612)

Additional reagents and equipment for preparing Matrigel ( protocol 5 ), culturing hESC (Lin and Talbot, ; unit 1.5 ), preparing standard curve ( protocol 3 ), counting cells using a hemacytometer ( protocol 2 ), and MTT assay ( protocol 5 )

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Figures

Figure 1.C1.1 Standard curve for determining mNSC concentration. This curve was produced by averaging three experiments performed on different days using different batches of cells. Eight cell counts were obtained for dilutions 1:2, 1:4, 1:8, and 1:16 using a hemacytometer. Percent transmittance readings were obtained for the undiluted sample, as well as dilutions 1:2 through 1:128. The micrograph (insert) shows mNSC in suspension.

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Figure 1.C1.2 MTT plate design. Single, double, or triple replicates can be carried out. The top controls (CN) are used to generate the coefficient of variation for plating accuracy, and the bottom controls are used to assess the possibility of a vapor effect. The numbers 1 to 6 represent low to high doses plated. Letters A, B, etc. designate different test groups. In this design, six different chemicals can be tested in duplicate on one plate.

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Figure 1.C1.3 Dose‐response curve showing the effect of phenol on mNSC. The x axis shows the doses tested, and the y axis shows the percent of the control ( n = 3). IC₅₀ = inhibitory concentration at 50%, LOAEL = lowest observed adverse effect level, NOAEL = no observable adverse effect level. * = p < 0.05.

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Figure 1.C1.4 The NIH Assay Guidance Wiki generates graphical data to check for edge effects and drift in the MTT plates. The validation experiment was carried out according to (Assay Validation: Plate Uniformity and Signal Variability), and no edge effects or drift were seen. (A ) Day 1 Plate 1. (B ) Day 1 Plate 2. (C ) Day 2 Plate 1. (D ) Day 2 Plate 2. These figures depict absorbance readings obtained for the three doses plotted as a function of column number. Different colors depict different doses: green, low dose/high signal; pink, mid dose/mid signal; blue, high dose/low signal. The readings from eight wells of one column of the plate are clustered together; for example, 1 to 8 on the x axis corresponds to eight wells in column 1. All the wells in each column had the same dose of chemicals.

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Figure 1.C1.5 Phenol dose‐response curve using ROCKi to generate single cells. The x axis shows the doses tested, and the y axis shows the percent of the control ( n = 3). IC₅₀ = inhibitory concentration at 50% and the NOAEL = no observable adverse effect level. ** = p ≤ 0.01. The LOAEL is between 3.19 × 10⁻⁵ and 9.37 × 10⁻⁵ M.

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Figure 1.C1.6 Standard curve for hESC. This curve was generated by obtaining eight cell counts for dilutions of 1:2, 1:4, 1:8, and 1:16 using a hemacytometer and obtaining percent transmittance readings for an undiluted sample as well as dilutions 1:2 through 1:128. The micrograph (insert) shows hESC in a uniform small‐colony suspension.

View Image

Figure 1.C1.7 Phenol dose‐response curve using the small‐colony method. The x axis shows the doses tested, and the y axis shows the percent of the control ( n = 3). IC₅₀ = inhibitory concentration at 50%, LOAEL = lowest observed adverse effect level, NOAEL = no observable adverse effect level. * = p < 0.05.

View Image

Figure 1.C1.8 The NIH Assay Guidance Wiki generates graphical data to check for edge effects and drift in the MTT plates. The validation experiment was carried out according to (Assay Validation: Plate Uniformity and Signal Variability), and no edge effects or drift were seen. (A ) Day 1 Plate 1. (B ) Day 1 Plate 2. (C ) Day 2 Plate 1. (D ) Day 2 Plate 2. These figures depict absorbance readings obtained for the three doses plotted as a function of column number. Different colors depict different doses: green, low dose/high signal; pink, mid dose/mid signal; blue, high dose/low signal. The readings from eight wells of one column of the plate are clustered together—for example, 1 to 8 on x axis corresponds to eight wells in column 1. All the wells in each column had the same dose of chemical.

View Image

Figure 1.C1.9 Variation in the MTT assay results when flicking versus swirling is used to keep cells in suspension. 5‐fluorouracil treatment of hESC is shown here where (A ) represents the flicking of cell suspension in a 15‐ml conical tube before plating each well with cells and (B ) represents swirling of the cell suspension in a 10‐ml glass beaker before plating each well with cells. The colored bars show quadruplicates for each dose. CN = control.

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Videos

Literature Cited

	Berridge, M.V., Tan, A.S., McCoy, K.D., and Wang, R. 1996. The biochemical and cellular basis of cell proliferation assays that use tetrazolium salts. Biochemica 4:14‐19.
	Blein, O., Ronot, X., and Adolphe, M. 1991. Cross contamination associated with the use of multiwell culture plates for cytotoxicity assessment of volatile chemicals. Cytotechnology 6:79‐82.
	Freshney, R.I. 2006. Basic principles of cell culture. In Culture of Cells for Tissue Engineering (R.I. Freshney and G. Vunjak‐Novakovic, eds.) pp. 3‐22. John Wiley & Sons, Hoboken, N.J.
	Fujimura, M., Usuki, F., Kawamura, M., and Izumo, S. 2011. Inhibition of the Rho/ROCK pathway prevents neuronal degeneration in vitro and in vivo following methylmercury exposure. Toxicol. Appl. Pharmacol. 250:1‐9.
	Grandjean, P., Bellinger, D., Bergman, A., Cordier, S., Davey‐Smith, G., Eskenazi, B., Gee, D., Gray, K., Hanson, M., van den Hazel, P., Heindel, J.J., Heinzow, B., Hertz‐Picciotto, I., Hu, H., Huang, T.T., Jensen, T.K., Landrigan, P.J., McMillen, I.C., Murata, K., Ritz, B., Schoeters, G., Skakkebaek, N.E., Skerfving, S., and Weihe, P. 2008. The faroes statement: Human health effects of developmental exposure to chemicals in our environment. Basic Clin. Pharmacol. Toxicol. 102:73‐75.
	Lin, S. and Talbot, P. 2011. Methods for culturing mouse and human embryonic stem cells. Methods Mol. Biol. 690:31‐56.
	Lin, S., Fonteno, S., Weng, J.H., and Talbot, P. 2010. Comparison of the toxicity of smoke from conventional and harm reduction cigarettes using human embryonic stem cells. Toxicol. Sci. 118:202‐212.
	Mossman, B.T. 1983. In vitro approaches for determining mechanisms of toxicity and carcinogenicity by asbestos in the gastrointestinal and respiratory tracts. Environ. Health Perspect. 53:155‐161.
	Ohgushi, M., Matsumura, M., Eiraku, M., Murakami, K., Aramaki, T., Nishiyama, A., Muguruma, K., Nakano, T., Suga, H., Ueno, M., Ishizaki, T., Suemori, H., Narumiya, S., Niwa, H., and Sasai, Y. 2010. Molecular pathway and cell state responsible for dissociation‐induced apoptosis in human pluripotent stem cells. Cell Stem Cell 7:225‐239.
	Parker, M.A., Anderson, J.K., Corliss, D.A., Abraria, V.E., Sidman, R.L., Park, K.I., Teng, Y.D., Cotanche, D.A., and Snyder, E.Y. 2005. Expression profile of an operationally‐defined neural stem cell clone. Exp. Neurol. 194:320‐332.
	Talbot, P. and Lin, S. 2011. Mouse and human embryonic stem cells: Can they improve human health by preventing disease? Curr. Top. Med. Chem. 11:1638‐1652.
	Watanabe, K., Ueno, M., Kamiya, D., Nishiyama, A., Matsumura, M., Wataya, T., Takahashi, J.B., Nishikawa, S., Muguruma, K., and Sasai, Y. 2007. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat. Biotechnol. 25:681‐686.
Internet Resources
	http://assay.nih.gov/assay/index.php/Assay_Validation_2011
	Assay validation Wiki.