Immunofluorescence labeling of cell surface antigens in Dictyostelium
© Vernay and Cosson; licensee BioMed Central Ltd. 2013
Received: 8 May 2013
Accepted: 9 August 2013
Published: 12 August 2013
Immunolocalization of cellular antigens typically requires fixation and permeabilization of cells, prior to incubation with antibodies.
Assessing a test protein abundantly present at the cell surface of Dictyostelium cells, we show that in fixed cells, permeabilization extracts almost completely this cell surface antigen. The extent of this artifact is variable depending on the procedure used for labeling and permeabilization, as well as on the antigen considered.
An optimized protocol for labeling both surface and intracellular antigens without significant loss of labeling is proposed.
In order to detect the presence of a protein in eukaryotic cells, and to determine its intracellular localization, it is common to label cells with specific fluorescent antibodies following cell fixation and permeabilization. Permeabilization must disrupt the cell membranes sufficiently to allow the passage of antibodies, while preserving the structure and protein composition of these same membranes. The problem is exacerbated at the level of the plasma membrane, which is the cellular membrane most exposed to solvents or detergents used to permeabilize cells.
Dictyostelium discoideum is a soil amoeba frequently used to study cell biology, in particular cell motility, endocytosis, cell adhesion or phagocytosis. For many of these studies it is critical to determine if membrane proteins implicated in these processes are located in intracellular compartments or exposed at the cell surface. Protocols used to permeabilize and stain Dictyostelium cells are fundamentally similar to those used with mammalian cells, with the caveat that Dictyostelium membranes can be more resistant to mild permeabilizing detergents like saponin.
In the course of our studies, we observed that different immunofluorescence protocols detected very different levels of proteins at the cell surface. In this study we show that permeabilization procedures remove a large amount of cell surface antigens. We also propose an optimal procedure to label both the cell surface and intracellular compartments.
Cells and reagents
Dictyostelium discoideum DH1-10 cells were grown at 21°C in HL5 medium (14.3 g/L Bactopeptone, 7.15 g/L Yeast Extract, 18 g/L Maltose monohydrate, 3.6 mM Na2HPO4.2H2O and 3.6 mM KH2PO4). Paraformaldehyde was purchased from by AppliChem, Saponin from Sigma and Triton X-100 was from Fluka.
The plasmid allowing expression of a fusion protein composed of the csA extracellular domain fused to the transmembrane domain of SibA and a short cytoplasmic domain (RRRSMAAA) was transfected in DH1-10 cells by electroporation. Transfected cells were then selected and grown in HL5 medium supplemented by G418 (10 μg/mL). For simplicity this fusion protein is referred to here as csA-SA. To detect csA-SA we used a mouse monoclonal antibody (41-71-21) directed to the csA extracellular domain. When indicated, p23, p25 and p80 membrane proteins were detected using H194, H72, and H161 mouse monoclonal antibodies. The unidentified H36 surface antigen recognized by the H36 monoclonal antibody was also described previously.
For all immunofluorescence procedures, 106Dictyostelium cells expressing csA-SA were allowed to attach to a 22×22 mm glass coverslip for 10 minutes at room temperature in 2 mM Na2HPO4, 14.7 mM KH2PO4, pH6.0 supplemented with 0.5% HL5, 100 mM sorbitol, and 100 μM CaCl2. This buffer allows optimal attachment of Dictyostelium cells to their substrate, while preserving optimally their general organization. Cells were then fixed for 10 minutes at room temperature in PBS containing 4% paraformaldehyde, then washed in PBS containing 20 mM NH4Cl, and in PBS containing 0.2% BSA (PBS-BSA).
In the immunofluorescence procedure referred to as “Classical”, cells were then washed twice in PBS, permeabilized in methanol at −20°C for 2 seconds, washed twice in PBS and once in PBS-BSA. When indicated, methanol was replaced with Triton X-100 (0.07% in PBS for 2 minutes at room temperature) or with saponin (0.2% in PBS for 10 minutes). Permeabilized cells were incubated with a mouse anti-csA antibody in PBS-BSA for 1 hour, washed twice in PBS-BSA, incubated for 1 hour with an Alexa-488-coupled anti-mouse immunoglobulin antibody in PBS-BSA, washed twice in PBS-BSA, once in PBS and mounted in Möwiol. Cells were visualized using a LSM700 confocal microscope (Zeiss). In each experiment, pictures from different samples were taken consecutively using identical settings.
In the procedure referred to as “Surface labeling”, non-permeabilized fixed cells were incubated with an anti-csA antibody in PBS-BSA for 1 hour, washed twice in PBS-BSA, incubated 1 hour with an Alexa-488-coupled anti-mouse antibody diluted in PBS-BSA. Finally, cells were washed twice in PBS-BSA, once in PBS and mounted in Möwiol.
In the procedure referred to as “Two-step” the surface of fixed cells was labeled as described above in the “Surface labeling” procedure. After surface labeling, cells were fixed again in paraformaldehyde, washed in PBS-NH4Cl, twice in PBS-BSA, twice in PBS before permeabilization in methanol at −20°C. Permeabilized cells were rinsed twice in PBS and once in PBS-BSA. Intracellular csA was then labeled for 1 hour with a mouse anti-csA antibody diluted in PBS-BSA, washed twice in PBS-BSA and revealed using an Alexa-488-coupled anti-mouse antibody. Finally, cells were washed twice in PBS-BSA, once in PBS and mounted in Möwiol.
Findings and discussion
In summary, we tested here three distinct procedures to permeabilize fixed cells prior to immunofluorescence staining: methanol, triton X-100 and saponin. All three methods resulted in a marked loss of cell surface labeling of the csA-SA protein. The csA-SA protein likely represents an extreme case since it is anchored to the cell membrane only by one transmembrane domain followed by a short cytoplasmic domain. When other surface proteins were tested, some (p23, p25, H36) were also largely extracted from the cell surface although they remained detectable. On the contrary, p80, maybe due to its three transmembrane domains, was not detectably extracted from the cell surface upon permeabilization.
These results suggest that when assessing the surface localization of a protein by immunofluorescence, it is best to compare results obtained using several alternative protocols in order to ascertain that no loss of labeling is caused by the permeabilization procedure. Ideally, a surface immunofluorescence of non-permeabilized cells should be performed. In some situations, it will be difficult to detect reliably a protein of interest at the cell surface, for example if no antibodies directed to the extracellular domain of the protein are available. It may then be necessary to define the most adequate compromise to perform immunofluorescence detection: one option is to use mild detergents like saponin to reduce the amount of protein lost from the cell surface upon permeabilization. Using very low concentrations of detergents may be an alternative approach, and sufficient permeabilization may even be achieved simply by paraformaldehyde fixation with no further permeabilization. It should however be kept in mind that very mild permeabilization procedures may result in incomplete permeabilization of some cellular membranes, as shown previously for saponin permeabilization in Dictyostelium. Use of alternative methodological approaches (e.g. cell surface biotinylation followed by biochemical analysis or expression of GFP-tagged proteins in live cells) not sensitive to the same type of artifacts may be necessary to detect and quantify unambiguously the presence of a protein at the cell surface.
This research was supported by a grant from the Swiss National Science Foundation (number 31003A-135789 to P.C.).
- Cosson P, Soldati T: Eat, kill or die: when amoeba meets bacteria. Curr Opin Microbiol. 2008, 11: 271-276. 10.1016/j.mib.2008.05.005.PubMedView ArticleGoogle Scholar
- Mercanti V, Cosson P: Resistance of Dictyostelium discoideum membranes to saponin permeabilization. BMC Res Notes. 2010, 3: 120-10.1186/1756-0500-3-120.PubMedPubMed CentralView ArticleGoogle Scholar
- Cornillon S, et al: Phg1p is a nine-transmembrane protein superfamily member involved in dictyostelium adhesion and phagocytosis. J Biol Chem. 2000, 275: 34287-34292.PubMedView ArticleGoogle Scholar
- Bertholdt G, et al: Carbohydrate and other epitopes of the contact site A glycoprotein of Dictyostelium discoideum as characterized by monoclonal antibodies. Cell Differ. 1985, 16: 187-202.PubMedView ArticleGoogle Scholar
- Ravanel K, et al: Membrane sorting in the endocytic and phagocytic pathway of Dictyostelium discoideum. Eur J Cell Biol. 2001, 80: 754-764. 10.1078/0171-9335-00215.PubMedView ArticleGoogle Scholar
- Mercanti V, et al: Selective membrane exclusion in phagocytic and macropinocytic cups. J Cell Sci. 2006, 119: 4079-4087. 10.1242/jcs.03190.PubMedView ArticleGoogle Scholar
- Smith EW, et al: Effect of starvation on the endocytic pathway in Dictyostelium cells. Eukaryotic cell. 2010, 9: 387-392. 10.1128/EC.00285-09.PubMedPubMed CentralView ArticleGoogle Scholar
- Lamvik J, et al: Nonlabeled secondary antibodies augment/maintain the binding of primary, specific antibodies to cell membrane antigens. Cytometry. 2001, 45: 187-193. 10.1002/1097-0320(20011101)45:3<187::AID-CYTO1162>3.0.CO;2-7.PubMedView ArticleGoogle Scholar
- Goldenthal KL, et al: Postfixation detergent treatment for immunofluorescence suppresses localization of some integral membrane proteins. J Histochem Cytochem: Official J Histochem Soc. 1985, 33: 813-820. 10.1177/33.8.3894499.View ArticleGoogle Scholar
- Lecoeur H, et al: A cytofluorometric method for the simultaneous detection of both intracellular and surface antigens of apoptotic peripheral lymphocytes. J Immunol Methods. 1998, 217: 11-26. 10.1016/S0022-1759(98)00060-X.PubMedView ArticleGoogle Scholar
- Hinners I, et al: The orientation of membrane proteins determined in situ by immunofluorescence staining. Anal Biochem. 1999, 276: 1-7. 10.1006/abio.1999.4325.PubMedView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.