Live‐Cell Immunofluorescence Staining of Human Pluripotent Stem Cells
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- Abstract
- Table of Contents
- Materials
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- Literature Cited
Abstract
Antibodies are instrumental tools in stem cell identification, purification, and analysis. Most commonly, cell samples are either dissociated to obtain a single?cell suspension suitable for FACS analysis or cell sorting, or fixed in situ for immunostaining and fluorescence microscopy imaging. This unit describes an alternative method in which live adherent cells are stained and imaged in situ without the need for cell dissociation, fixation, or fluorescent reporter genes. This minimally invasive method is particularly useful for identification and distinction of fully and partially reprogrammed induced pluripotent stem cells (iPSCs). The unit also describes the use of mCD49e and hCD29 antibodies in live?cell (vital) imaging. mCD49e strongly stains mouse embryonic fibroblast (MEF) feeder cells in human pluripotent stem cell cultures, whereas hCD29 recognizes an antigen expressed on undifferentiated and many differentiated cells. A distinguishing feature of hCD29 in live?cell staining is that its antigen is precluded from detection wherever cells have formed tight epithelial junctions (e.g., in the center but not the periphery of pluripotent stem cell colonies) due to basolateral location. A non?fluorescent fixed?cell staining protocol is also provided for medium? to high?throughput quantification of stem cell experiments without an automated microscope. The discussion addresses technical limitations, pitfalls, troubleshooting, and potential applications, such as identification of emerging bona fide human iPSC colonies in reprogramming experiments. Curr. Protoc. Stem Cell Biol. 19:1C.12.1?1C.12.14. © 2011 by John Wiley & Sons, Inc.
Keywords: human embryonic stem cells; human pluripotent stem cells; human induced pluripotent stem cells; reprogramming; imaging; immunofluorescence staining; vital staining
Table of Contents
- Introduction
- Basic Protocol 1: Live Immunofluorescence Staining of Adherent Pluripotent Stem Cells
- Basic Protocol 2: Automated Fluorescence Microscopy of Live‐Stained Stem Cells
- Basic Protocol 3: Image Processing and Analysis
- Alternate Protocol 1: Non‐Fluorescent Chromogenic Development of Live and Fixed Cell Antibody Stainings
- Reagents and Solutions
- Commentary
- Literature Cited
- Figures
- Tables
Materials
Basic Protocol 1: Live Immunofluorescence Staining of Adherent Pluripotent Stem Cells
Materials
Basic Protocol 2: Automated Fluorescence Microscopy of Live‐Stained Stem Cells
Materials
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Figures
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Figure 1.C1.1 (A ) A grayscale flatfield image (single 256 × 256–pixel frame). (B ) 2 × 2 flatfield correction frames. (C ) A human pluripotent stem cell colony (2 × 2 montage). (D ) The same montage in (C) after flatfield correction using the flatfield correction image in (B). (E ) Pseudocolored multi‐frame montage of live‐stained and imaged human embryonic stem cells grown on mouse embryonic fibroblasts. Red, mCD49e; green, hCD29 (stains differentiated, mesenchymal human cells and incompletely epithelialized periphery of stem cell colonies); blue, TRA‐1‐60 (only expressed by undifferentiated stem cells). (F ) A 10 × 1 montage flatfield correction image (pseudocolored 3D plot; purple = brightest, yellow = darkest). Note that each frame has a unique shape and height due to positional effects (e.g., different optical properties across the culture dish, well walls, etc.). View Image -
Figure 1.C1.2 (A ) Brightfield image of two human pluripotent stem cell colonies grown on mouse embryonic fibroblast feeder cells. (B ‐C ) The same colonies are shown after live‐cell staining with hCD29 (green; B) and post‐fixation staining with SSEA4 (C). Arrowhead in (A, B) denotes human mesenchymal cells that differentiated and migrated away from the nearby human pluripotent stem cell colony. Note that these CD29+ cells (B) cannot be easily identified using phase contrast alone (A). (D ) In the merge of (B) and (C), live‐cell staining allows human cells to be detected among mouse embryonic fibroblasts, which would be difficult, if not impossible, using standard brightfield microscopy. (E ) HRP‐based chromogenic stain (hCD29, dark‐purple precipitate; stained post‐fixation) of human pluripotent stem cell colonies. (F ) HRP‐based stain (hCD29, dark purple precipitate; live‐staining followed by fixation) of human pluripotent stem cell colonies. (E) and (F) were counterstained using a standard alkaline phosphatase staining kit to reveal expression of the pluripotent stem cell marker APLP. Note that while hCD29 is expressed by all human pluripotent stem cells throughout each colony (E), this antibody fails to stain live cells in the epithelialized centers of undifferentiated human pluripotent stem cell colonies (F). View Image
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Literature Cited
Literature Cited | |
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