Immunophenotyping Using a Laser Scanning Cytometer

Attila Tárnok1, Andreas O.H. Gerstner1

1 University of Leipzig, Leipzig
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 6.13
DOI:  10.1002/0471142956.cy0613s23
Online Posting Date:  February, 2003
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Abstract

Three‐ and four‐color immunophenotyping is routine in traditional flow cytometry, as is measurement of cell proliferation, but there are drawbacks. The techniques cannot analyze cell morphology or permit restaining of cells of interest. This unit describes a slide‐based method of immunophenotyping using a laser scanning cytometer. In general, many assays originally developed for flow can be adapted to LSC. Although the speed of analysis is comparatively slow, LSC has the advantage that cells are not lost. Considerable additional information can be obtained by morphological examination and/or by further staining because specimens can be repeatedly analyzed and archived. The method has potential to become a powerful tool in clinical diagnosis.

     
 
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Table of Contents

  • Basic Protocol 1: Immunophenotyping of Peripheral Blood Leukocytes
  • Alternate Protocol 1: Four‐Color Immunophenotyping Without Nuclear Staining
  • Support Protocol 1: Setting Up a Laser Scanning Cytometer
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Immunophenotyping of Peripheral Blood Leukocytes

  Materials
  • Peripheral blood sample (≤500 µl) collected in 1.5‐ml syringe containing EDTA (Kabe Labortechnik)
  • Monoclonal antibodies (e.g., CD3, CD4, CD8, CD14, CD45 from BD Biosciences, Caltag Laboratories, Beckman Coulter, DakoCytomation, or other commercial sources) conjugated to fluorescein isothiocyanate (FITC), phycoerythrin (PE), and allophycocyanin (APC)
  • Lysing solution: ammonium chloride lysing solution ( appendix 2A) or commercial equivalent (e.g., FACS lysing solution; BD Biosciences)
  • PBS ( appendix 2A), room temperature and 4°C
  • 0.5% (w/v) paraformaldehyde in PBS ( appendix 2A)
  • PBS/BSA: 0.5% (w/v) BSA/0.016% (w/v) sodium azide in PBS ( appendix 2A), pH 7.40
  • Acetone, 4°C
  • Fluorescence mounting medium (e.g., DakoCytomation)
  • 5 µg/ml 7‐aminoactinomycin D (7‐AAD; Sigma) in PBS/BSA
  • Hematoxylin and eosin (H & E): purchase a commercial kit or see Taboas and Ceremsak ( )
  • 70%, 80%, 90%, and 100% (v/v) ethanol
  • Xylene (e.g., E. Merck)
  • Permanent mounting medium for cytological slides (e.g., Eukitt; Kindler)
  • 1.5‐ml microcentrifuge tubes or 12 × 75–mm polystyrene tubes (e.g., Falcon)
  • Grease pencil to mark slides
  • Uncoated glass microscope slides
  • Micropipettor and plastic tips
  • Laminar flow chamber
  • Coplin jars
  • Humidified chamber for incubating slides
  • 24 × 24–mm coverslips
  • Laser scanning cytometer (LSC; CompuCyte) equipped with argon (Ar) and helium/neon (HeNe) laser; standard filter settings for the measurement of FITC, PE, APC, and 7‐AAD fluorescence; and WinCyte software
  • Additional reagents and equipment for setting up LSC (see protocol 3) and H & E staining (Taboas and Ceremsak, )
NOTE: Common EDTA syringes are available in sizes >1.5 ml and provide far more material than is required for the assay. Subdivide the EDTA solution from the syringe into small neutral syringes and reduce the volume of blood drawn accordingly.

Alternate Protocol 1: Four‐Color Immunophenotyping Without Nuclear Staining

  • PE‐Cy5‐conjugated monoclonal antibody

Support Protocol 1: Setting Up a Laser Scanning Cytometer

  Materials
  • Laser scanning cytometer (LSC; CompuCyte) equipped with argon (Ar) and helium/neon (HeNe) laser; standard filter settings for the measurement of FITC, PE, APC, and 7‐AAD fluorescence; and WinCyte software
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Figures

Videos

Literature Cited

Literature Cited
   Bajaj, S., Welsh, J.B., Leif, R.C., and Price, J.H. 2000. Ultra‐rare‐event detection performance of a custom scanning cytometer on a model preparation of fetal nRBCs. Cytometry 39:285‐294.
   Bedner, E., Halicka, H.D., Cheng, W., Salomon, T., Deptala, A., Gorczyca, W., Melamed, M.R., and Darzynkiewicz, Z. 1999. High affinity binding of fluorescein isothiocyanate to eosinophils detected by laser scanning cytometry: A potential source of error in analysis of blood samples utilizing fluorescein‐conjugated reagents in flow cytometry. Cytometry 36:77‐82.
   Chase, E.S. and Hoffman, R.A. 1998. Resolution of dimly fluorescent particles: A practical measure of fluorescence sensitivity. Cytometry 33:267‐279.
   Gerstner, A.O.H., Laffers, W., Bootz, F., and Tárnok, A. 2000. Immunophenotyping of peripheral blood leukocytes by laser scanning cytometry. J. Immunol. Methods 246:175‐185.
   Gerstner, A.O.H., Lenz, D., Laffers, W., Hoffman, R.A., Steinbrecher, M., Bootz, F., Tárnok, A. 2002. Near‐infrared dyes for six color immunophenotyping by LSC. Cytometry 48:115‐123.
   Kamentsky, L.A. and Kamentsky, L.D. 1991. Microscope‐based multiparameter laser scanning cytometer yielding data comparable to flow cytometry data. Cytometry 12:381‐387.
   Kamentsky, L.A., Burger, D.E., Gershman, R.J., Kamentsky, L.D., and Luther, E. 1997. Slide‐based laser scanning cytometry. Acta Cytol. 41:123‐143.
   Loborg, H., Linden, E., Lonn, A., Skoglund, P., and Rundquist, I. 1995. High affinity binding of 7‐aminoactinomycin D and 4′,6‐diamidino‐2‐phenylindole to human neutrophilic granulocytes and lymphocytes. Cytometry 20:296‐306.
   Roederer, M., Kantor, A.B., Parks, D.R., and Herzenberg, L.A. 1996. Cy7PE and Cy7APC: Bright new probes for immunofluorescence. Cytometry 24:191‐197.
   Schmid, I., Cole, S.W., Zack, J.A., and Giorgi, J.V. 2000. Measurement of lymphocyte subset proliferation by three‐color immunofluorescence and DNA flow cytometry. J. Immunol. Methods 235:121‐131.
   Taboas, J.O. and Ceremsak, R.J. 1967. A rapid hematoxylin and eosin stain. Tech. Bull. Regist. Med. Technol. 37:119‐120.
   Tárnok, A. and Gerstner, A.O.H. 2002. Clinical applications of laser scanning cytometry. Cytometry 50:133‐143.
   Wood, J.C. 1998. Fundamental flow cytometer properties governing sensitivity and resolution. Cytometry 33:260‐266.
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