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

Rachel Z. Behar1, Vasundhra Bahl1, Yuhuan Wang2, Jo‐Hao Weng3, Sabrina C. Lin2, Prue Talbot2

1 These authors contributed equally to this work., 2 Department of Cell Biology and Neuroscience, University of California, Riverside, California, 3 Cell, Molecular and Developmental Biology Graduate Program, University of California, Riverside, California
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1C.13
DOI:  10.1002/9780470151808.sc01c13s23
Online Posting Date:  November, 2012
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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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Table of Contents

  • 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

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.
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