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Labeling Lipids for Imaging in Live Cells Carsten Schultz, Anne B. Neef, Theodorus W. Gadella, Jr and Joachim Goedhart Cold Spring Harb Protoc 2010; doi: 10.1101/pdb.prot5459 Email Alerting Service Subject Categories
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Protocol
Labeling Lipids for Imaging in Live Cells Carsten Schultz, Anne B. Neef, Theodorus W. Gadella Jr, and Joachim Goedhart INTRODUCTION Fluorescently tagged lipid-binding domains have become a popular tool to image lipids that are involved in intracellular signaling processes. The readout usually involves the translocation of the lipidbinding domain from the cytosol or nucleosol to the membrane of interest, or vice versa. Unfortunately, this method seems to work predominantly for lipids in the plasma membrane, whereas lipids such as phosphatidylinositol 4,5-bisphosphate (PIP2) are not recognized in the membranes of the endoplasmic reticulum or the Golgi. Very recently, we developed an alternative way of localizing a lipid of interest by fluorescent labeling of minimally modified lipid derivatives using a single specific chemical reaction. For lipid location analyses, the method is used in fixed cells. However, for studying lipid dynamics, specific labeling in living cells is also possible. This protocol describes how to directly label lipids for imaging in living cells.
RELATED INFORMATION For more information, see Neef and Schultz (2009) and Imaging Lipids in Living Cells (Schultz et al. 2010a). Protocols are also available for Transfection of Cells with DNA Encoding a Visible Fluorescent Protein-Tagged Lipid-Binding Domain (Schultz et al. 2010b) and Labeling Lipids for Imaging in Fixed Cells (Schultz et al. 2010c).
MATERIALS CAUTIONS AND RECIPES: Please see Appendices for appropriate handling of materials marked with , and
recipes for reagents marked with .
Reagents Cells of interest (e.g., RAW microphages, HeLa cells) with appropriate culture medium Dulbecco’s PBS for lipid labeling (DPBS) (prewarmed to 37°C) Fetal bovine serum (FBS) (10% prepared in HEPES buffer and prewarmed to 37°C) Imaging buffer (prewarmed to 37°C) Lipid mix B Pluronic F-127 (5% prepared in DMSO) Staining mix C (prewarmed to 37°C)
Equipment Cell culture chambers (35-mm) containing glass coverslips (MatTek Corporation) Incubator preset to 37°C and 5% CO2 Micropipettor and tips Microscope, fluorescence (with 405-nm laser line) Shaker (in the dark and at 37°C)
Adapted from Live Cell Imaging, 2nd edition (ed. Goldman et al.). CSHL Press, Cold Spring Harbor, NY, USA, 2010. Cite as: Cold Spring Harb Protoc; 2010; doi:10.1101/pdb.prot5459 © 2010 Cold Spring Harbor Laboratory Press
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METHOD 1. Grow the cells in 35-mm cell culture chambers in appropriate medium for 1 d at 37°C and
5% CO2. 2. Wash the cells twice with DPBS prewarmed to 37°C. 3. Add 2 mL of prewarmed 10% FBS in HEPES buffer to cover the cells. 4. Supplement the cells with 5 µL of freshly prepared lipid mix B to a final lipid concentration of
10 µM. Add an equal amount of freshly prepared 5% Pluronic F-127 in DMSO to the negative control dish. 5. Incubate the cells for 1 h at 37°C and 5% CO2. 6. Rinse the cells twice with DPBS prewarmed to 37°C. 7. Add 2 mL of freshly prepared staining mix C prewarmed to 37°C. 8. Tightly seal the chambers. Incubate the cells on a shaker in the dark for 3 h at 37°C. 9. Remove the staining mix. Wash the cells in DPBS prewarmed to 37°C three times for 1 min each. 10. Cover the cells with imaging buffer. Image the cells by fluorescence microscopy (excitation
at 405 nm, 505/30 bandpass filter for emission). See Figure 1 for sample results.
FIGURE 1. Labeling of cyclooctyne-bearing lipids with fluorogenic 3-azido-7-(diethylamino)coumarin in living cells. (A) Coumarin channel; (B) differential interference contrast (DIC); (C) negative control cells without lipid added; coumarin channel; (D) negative control cells, DIC.
REFERENCES Neef AB, Schultz C. 2009. Selective fluorescence labeling of lipids in living cells. Angew Chem Int Ed 48: 1498–1500. Schultz C, Neef AB, Gadella TW Jr, Goedhart J. 2010a. Imaging lipids in living cells. Cold Spring Harb Protoc (this issue). doi: 10.1101/ pdb.top83. Schultz C, Neef AB, Gadella TW Jr, Goedhart J. 2010b. Transfection of
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cells with DNA encoding a visible fluorescent protein-tagged lipid-binding domain. Cold Spring Harb Protoc (this issue). doi: 10.1101/pdb.prot5457. Schultz C, Neef AB, Gadella TW Jr, Goedhart J. 2010c. Labeling lipids for imaging in fixed cells. Cold Spring Harb Protoc (this issue). doi: 10.1101/pdb.prot5458.
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