Methods and Applications of Laser‐Enabled Analysis and Processing (LEAP)

Nan Lin1, Jennifer R. Cresswell1, Genova A. Richardson1, Mark A. Gerber1, Kevin J. Kayser1

1 Cell Sciences and Development, SAFC Biosciences, St. Louis, Missouri
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 2.14
DOI:  10.1002/0471142956.cy0214s43
Online Posting Date:  January, 2008
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Abstract

The LEAP (Laser‐Enabled Analysis and Processing) platform combines in situ imaging with laser manipulation to efficiently identify, purify, and monitor expansion of high secreting clones. It also allows for rapid analysis of cell population heterogeneity. This unit describes the LEAP instrumentation as well as basic and alternate protocols of the major applications in recombinant human or humanized IgG expression. The protocols include fluorescent cell counting, secreted recombinant IgG capture and detection, and IgG‐secreting clone selection by laser processing. Curr. Protocol. Cytom. 43:2.14.1‐2.14.27. © 2008 by John Wiley & Sons, Inc.

Keywords: Laser‐Enabled Analysis and Processing (LEAP); recombinant IgG production; high‐producing cell line generation; in situ detection; secretion heterogeneity

     
 
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Table of Contents

  • The Leap System at a Glance
  • Strategic Planning
  • Basic Protocol 1: Fluorescent Cell Counting
  • Alternate Protocol 1: Fluorescent Cell Counting for Adherent Cells
  • Alternate Protocol 2: Two‐Channel Fluorescent Cell Counting
  • Alternate Protocol 3: Using Two‐Channel Fluorescent Cell Counting for Transfection Efficiency Assessment
  • Secreted Recombinant IgG Capture and Detection
  • Basic Protocol 2: IgG Capture and Detection for Suspension Cells
  • Alternate Protocol 4: IgG Capture and Detection for Adherent Cells
  • Alternate Protocol 5: IgG Capture and Detection in 96‐Well Plates
  • Alternate Protocol 6: Transient IgG Expression Assessment
  • Basic Protocol 3: Single‐Cell Cloning
  • Support Protocol 1: Laser Calibration
  • Support Protocol 2: Laser Parameter Optimization (Kill Curves)
  • Support Protocol 3: Optocleaning
  • Support Protocol 4: Expansion Condition Screen
  • Alternate Protocol 7: Population Enrichment
  • Alternate Protocol 8: Cell Population Purification Based on Other Markers
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Fluorescent Cell Counting

  Materials
  • Cell suspension of cell line(s) in appropriate culture medium
  • Appropriate fluorescent cell dye (see Table 2.14.3 for a list of possible dyes)
  • Culture medium supplemented appropriately, prewarmed to 37°C
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Centrifuge capable of 100 × g and rotor for microplates
  • LEAP system (see )
  • C‐lect 384‐well plates, tissue culture treated (Cyntellect, no. 10010003822)
  • Electronic single‐ and multi‐channel repeater pipets with speed control—e.g., EDP‐3 Advanced electronic pipette (Rainin)
    Table 2.4.3   MaterialsFluorescent Dyes Commonly Used for Simultaneous Two‐Color Cell Imaging

    Dye 1 Dye 2/fluorecent reporter protein Application
    CellTracker Orange Green Fluorescent Protein Transfection Efficiency
    CellTracker Green or Calcein AM Red Fluorescent Protein Transfection Efficiency
    CellTracker Orange Sytox Blue or Sytox Green Viability
    CellTracker Green or Calcein AM Propidium Iodide Viability
    CellTracker Green or Calcein AM Sytox Blue Viability

Alternate Protocol 1: Fluorescent Cell Counting for Adherent Cells

  • Trypsin‐EDTA
  • Additional reagents and equipment for trypsinization of cells and counting cells using trypan blue exclusion ( appendix 3B)

Alternate Protocol 2: Two‐Channel Fluorescent Cell Counting

  • A second appropriate fluorescent cell dye (see Table 2.14.3 for a list of possible dyes)

Alternate Protocol 3: Using Two‐Channel Fluorescent Cell Counting for Transfection Efficiency Assessment

  • Expression vector for a fluorescent reporter protein
  • Additional reagents and equipment for transfection (e.g., lipid‐based, calcium phosphate, or electroporation; Ausubel et al., )

Basic Protocol 2: IgG Capture and Detection for Suspension Cells

  Materials
  • Cell suspension of IgG‐expressing cell line(s) and the corresponding parental cell line(s) in appropriate serum‐free culture medium (at least 5 × 105 viable cells/ml with viability ≥90%)
  • 5 mg/ml protein G (GE Health Care, no. 17061901) stock solution in sterile tissue‐culture grade H 2O
  • Serum‐free culture medium supplemented with appropriate selection reagent(s), prewarmed to 37°C
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • CellTracker Green (Invitrogen, no. C7025), 5 mM in DMSO
  • Detection antibody: R‐Phycoerythrin‐conjugated donkey anti–human IgG antibody – F(ab') 2 fragment (Jackson Laboratories, no. 709‐116‐149), 1 mg/ml in sterile tissue‐culture grade H 2O
  • C‐lect 384‐well plates, tissue culture treated (Cyntellect, no. 10010003822)
  • Electronic single‐channel and multi‐channel repeater pipets with speed control (e.g., EDP‐3 Advanced electronic pipet, Rainin)
  • Centrifuge capable of 51 × g and rotor for microplates
  • Humidified incubator at 37°C/5% CO 2
  • LEAP system (see )
  • Additional reagents and equipment for counting cells using trypan blue exclusion ( appendix 3B)

Alternate Protocol 4: IgG Capture and Detection for Adherent Cells

  • Trypsin‐EDTA
  • Additional reagents and equipment for trypsinization and counting cells using trypan blue exclusion ( appendix 3B)

Alternate Protocol 5: IgG Capture and Detection in 96‐Well Plates

  • C‐lect 96‐well plates (Cyntellect, no. 10010009601)

Alternate Protocol 6: Transient IgG Expression Assessment

  • Additional reagents and equipment for transfection (e.g., lipid‐based, calcium phosphate, or electroporation; Ausubel et al., ) and counting cells using trypan blue exclusion ( appendix 3B)

Basic Protocol 3: Single‐Cell Cloning

  Materials
  • Expansion medium supplemented with appropriate selection reagent(s), prewarmed
  • LEAP system (see )
  • Electronic single‐channel and multi‐channel repeater pipettes with speed control
  • Additional reagents and equipment for laser optimization ( protocol 11)

Support Protocol 1: Laser Calibration

  Materials
  • LEAP system (see )
  • Calibration target slide (Cyntellect)

Support Protocol 2: Laser Parameter Optimization (Kill Curves)

  Materials
  • Cell suspension of cell line of interest (≥5 × 106 cells/ml, ≥90% viable)
  • Serum‐free culture medium supplemented with appropriate selection reagent(s)
  • CellTracker Green (Invitrogen, Catalog Number C7025), 5 mM in DMSO
  • Phosphate‐buffered saline ( appendix 2A)
  • Growth medium appropriate to cells
  • Expansion medium supplemented with appropriate selection reagent(s), prewarmed
  • CellTracker Orange CMTMR (Invitrogen, no. C2927), 5 mM in DMSO
  • Sytox Blue (Invitrogen, no. S11348)
  • Electronic multi‐channel repeat pipettor with speed control
  • 25‐cm2 flasks
  • 37°C incubator
  • 15‐ml conical tube
  • C‐lect 384‐well plates (Cyntellect, no. 10010003822)
  • Additional reagents and equipment for counting cells using trypan blue exclusion ( appendix 3B)

Support Protocol 3: Optocleaning

  Additional Materials (also see previous protocols)
  • Culture medium (fresh or conditioned) and sera as needed for the cell lines
  • Additional reagents and equipment for counting cells using trypan blue exclusion ( appendix 3B)
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Figures

Videos

Literature Cited

Literature Cited
   Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K., (eds.) 2007. Current Protocols in Molecular Biology. John Wiley & Sons Hoboken, N.J.
   Clark, I.B., Hanania, E.G., Stevens, J., Gallina, M., Fieck, A., Brandes, R., Palsson, B.O., and Koller, M.R. 2006. Optoinjection for efficient targeted delivery of a broad range of compounds and macromolecules into diverse cell types. J. Biomed. Opt. 11:014034.
   Hanania, E.G., Fieck, A., Stevens, J., Bodzin, L.J., Palsson, B.Φ., and Koller, M.R. 2005. Automated in situ measurement of cell‐specific antibody secretion and laser‐mediated purification for rapid cloning of highly‐secreting producers. Biotechnol. Bioeng. 91:872‐876.
   Koller, M.R., Hanania, E.G., Stevens, J., Eisfeld, T.M., Sasaki, G.C., Fieck, A., and Palsson, B.Φ. 2004. High‐throughput laser‐mediated in situ cell purification with high purity and yield. Cytometry A 61:153‐161.
   Kulms, D. and Schwarz, T. 2000. Molecular mechanisms of UV‐induced apoptosis. Photodermatol. Photoimmunol. Photomed. 16:195‐201.
   Kulms, D., Zeise, E., Pöppelmann, B., and Schwarz, T. 2002. DNA damage, death receptor activation and reactive oxygen species contribute to ultraviolet radiation‐induced apoptosis in an essential and independent way. Oncogene 21:5844‐5851.
   Sambrook, J. and Russell, D.W. 2001. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
   Szaniszlo, P., Rose, W.A., Wang, N., Reece, L.M., Tsulaia, T.V., Hanania, E.G., Elferink, C.J., and Leary, J.F. 2006. Scanning cytometry with a LEAP: Laser‐enabled analysis and processing of live cells in situ. Cytometry A 69:641‐651.
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