Dynamic Thermoregulation of the Sample in Flow Cytometry

Steven W. Graves1, Robert C. Habbersett1, John P. Nolan1

1 Los Alamos National Laboratories, Los Alamos, New Mexico
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 1.18
DOI:  10.1002/0471142956.cy0118s20
Online Posting Date:  May, 2002
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Abstract

Fine control of temperature is an important capability for any analytical platform. A circulating water bath has been the traditional means of maintaining constant temperature in the sample chamber of a flow cytometer, but this approach does not permit rapid changes in sample temperature. This unit explains the use of Peltier modules for regulation of sample temperature. The heat pumping generated by the passage of current through properly matched semiconductors, known as the Peltier effect, makes it possible for these thermoelectric modules to both heat and cool. The authors describe the construction of a Peltier module based thermoregulation unit in step‐by‐step detail and present a demonstration of flow cytometry measurements as a function of temperature.

     
 
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Table of Contents

  • Basic Protocol 1: Flow Cytometry Measurements as a Function of Temperature
  • Support Protocol 1: Construction and Mounting of a Peltier Module–Based Thermoregulation Unit
  • Support Protocol 2: Calibrating the Inline Thermoregulation Unit
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Flow Cytometry Measurements as a Function of Temperature

  Materials
  • Sample
  • Flow cytometer with time‐stamp capability and equipped with a Peltier module–based thermoregulation unit (see protocol 2)
  • Flow cytometry analysis software capable of exporting the mean of a parameter as a function of time (e.g., IDYLK, National Flow Cytometry Resource, http://www.bdiv.lanl.gov/NFCR/; or FlowJo, TreeStar Software)
  • Spreadsheet software (e.g., Excel, Microsoft; or Quattro Pro, Corel)

Support Protocol 1: Construction and Mounting of a Peltier Module–Based Thermoregulation Unit

  Materials
  • Copper block machined to 4 × 4 × 0.75 cm
  • Titanium tubing (5‐cm length, 1/16‐in. o.d., 0.01‐in. i.d., Upchurch)
  • Silicone heat‐sink compound (e.g., Dow Corning 340, Dow Corning)
  • Tellurex Peltier module–based thermoregulation unit
  • Melcor heat sink, model HX8‐201
  • Plastic screws, washers, and clamps
  • Three red banana jacks
  • Three black banana jacks
  • 12 V fan (Papst)
  • Type J thermocouple (1/16‐in. o.d. and 6‐in. length, Omega Engineering)
  • Watlow Electric Manufacturing model F4 ramping process controller
  • 16‐G electrical wire
  • Crydom relay heat sinks, model HS‐2
  • Four Crydom model D1D12 solid‐state DC relays
  • Four Newark Electronics model MUR1520 rectifiers, ON semiconductor
  • 2 banana plug to banana plug black patch cords rated to 15 A
  • 2 banana plug to banana plug red patch cords rated to 15 A
  • Newark Electronics DC power supply, Tenma‐72‐6153
  • Flow cytometer
  • Low‐profile vented electronics case, model 94F4839 (Newark Electronics)
  • Flexible silicone tubing (1/16‐in. o.d. and 0.01‐in. i.d.)
  • PEEK tubing (0.030 in. × 1/16 in. × 200 cm, 912 µl; Upchurch)
  • 3‐way valve (Upchurch)
  • Programmable syringe pump connected to syringe

Support Protocol 2: Calibrating the Inline Thermoregulation Unit

  Materials
  • Microspheres
  • 50 mM 2‐[N‐morpholino]ethanesulfonic acid (MES) buffer, pH 6.5 (see recipe)
  • DNA oligomer ∼25 bases in length with a 5′ NH 3 group
  • 1‐ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide (EDAC)
  • 1× PBS buffer, pH 7 ( appendix 2A)
  • DNA oligomer complementary to the specific DNA oligomer and with a 5′ fluorescent group attached
  • Flow cytometer with time‐stamp capability and equipped with a Peltier module–based thermoregulation unit (see protocol 2)
  • Flow cytometry analysis software capable of exporting the mean of a parameter as a function of time (e.g., IDYLK, National Flow Cytometry Resource, http://www.bdiv.lanl.gov/NFCR/; or FlowJo, TreeStar Software)
  • Spreadsheet software
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Figures

Videos

Literature Cited

Literature Cited
   Cai, H., White, P.S., Torney, D., Deshpande, A., Wang, Z., Keller, R.A., Marrone, B., and Nolan, J.P., 2000. Flow cytometry‐based minisequencing: A new platform for high‐throughput single‐nucleotide polymorphism scoring. Genomics 66:135‐143.
   Graves, S.W., Habbersett, R.C., and Nolan, J.P. 2001. A dynamic inline sample thermoregulation unit for flow cytometry. Cytometry 43:23‐30.
   Kelley, K.A. 1991 Very early detection of changes associated with cellular activation using a modified flow cytometer. Cytometry 12:464‐468.
   Kettman, J. R., Davies, T., Chandler, D., Oliver, K.G., and Fulton, R.J. 1998. Classification and properties of 64 multiplexed microsphere sets. Cytometry 33:234‐243.
   McMurray, C.T. 1999. DNA secondary structure: A common and causative factor for expansion in human disease. Proc. Natl. Acad.Sci. U.S.A. 96:1823‐1825.
   Nolan, J.P. and Sklar, L.A. 1998. The emergence of flow cytometry for sensitive, real‐time measurements of molecular interactions. Nat.Biotechnol. 16:633‐638.
   Omann, G.M., Coppersmith, W., Finney, D.A., and Sklar, L.A. 1985. A convenient on‐line device for reagent addition, sample mixing, and temperature control of cell suspensions in flow cytometry. Cytometry 6:69‐73.
   Ririe, K.M., Rasmussen, R.P., and Wittwer, C.T. 1997. Product differentiation of DNA melting curves during the polymerase chain reaction. Anal. Biochem. 245:154‐160.
   Rowe, D.M. 1995. CRC Handbook of Thermoelectrics. CRC Press, Boca Raton, Florida.
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