Calibration of Flow Cytometry for Quantitative Quantum Dot Measurements

Rowena Mittal1, Marcel P. Bruchez1

1 Molecular Biosensor and Imaging Center, Carnegie Mellon University, Pittsburgh, Pennsylvania
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 6.26
DOI:  10.1002/0471142956.cy0626s49
Online Posting Date:  July, 2009
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Observations of quantum dot (QD)–labeled cells in biomedical research are mainly qualitative in nature, which limits the ability of researchers to compare results experiment to experiment and laboratory to laboratory. Labeled cells are useful in a range of in vitro and in vivo assays where tracking behavior of administered cells is integral for answering research questions in areas such as tissue engineering and stem cell therapy. Before the full potential of QD‐based toolsets can be realized, uptake of QDs by cells must be quantified and standardized. This unit describes a novel, simple method to assess the number of QDs per cell using flow cytometry and commercially available standards. This quick and easy method can be used to calibrate flow cytometry instruments and settings, and quantify QD uptake by cells for in vitro and in vivo experimentation for comparable results across QD conjugate types, cell types, research groups, lots of commercial QDs, and homemade QDs. Curr. Protoc. Cytom. 49:6.26.1‐6.26.7. © 2009 by John Wiley & Sons, Inc.

Keywords: quantitative; quantum dot; cell uptake; flow cytometry; number of quantum dots per cell; QD uptake

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Table of Contents

  • Introduction
  • Basic Protocol 1: Flow Cytometer Calibration
  • Basic Protocol 2: Quantitative Measurement of QD Uptake by Cells Using Calibration Curve
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Flow Cytometer Calibration

  • 0.1% bovine serum albumin (BSA)/0.05 M Tris‐phosphate buffered saline (TPS) (see recipe), pH 8.0
  • Mouse IgG–conjugated QDs (IgG QDs) with desired emission wavelength (Invitrogen)
  • Quantum Simply Cellular (QSC) anti‐mouse IgG beads (Bangs Laboratories, 8‐µm)
  • Mouse IgG (Sigma)
  • Reference standard beads for desired emission wavelength
  • 0.22‐µm filter unit (Nalgene)
  • Vortexer
  • 1.5‐ml microcentrifuge tubes with screw caps (Eppendorf)
  • Rotator
  • Flow cytometer with 488‐nm or 400‐nm laser and appropriate filter sets for the emission wavelength of the QD under study and analysis software (e.g., FlowJo)
  • Curve‐fitting software (e.g., Graphpad Prism)

Basic Protocol 2: Quantitative Measurement of QD Uptake by Cells Using Calibration Curve

  • Labeled cells with QDs of emission wavelength calibrated on flow cytometer
  • Unlabeled cells without QDs (negative control)
  • 1× phosphate buffered saline (PBS; appendix 2A)
  • Flow cytometry tubes
  • Flow cytometer
  • Flow cytometry data analysis software (e.g., FlowJo)
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Literature Cited

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