Use of the Intracellular Fluorescent Dye. CFSE to Monitor Lymphocyte Migration and Proliferation. Christopher R. Parish,1 Megan H. Glidden,1 Ben J. C. Quah,1 ...
Use of the Intracellular Fluorescent Dye CFSE to Monitor Lymphocyte Migration and Proliferation
UNIT 4.9
Christopher R. Parish,1 Megan H. Glidden,1 Ben J. C. Quah,1 and Hilary S. Warren1 1
Australian National University, Canberra, Australia
ABSTRACT The stable incorporation of the intracellular fluorescent dye 5-(and -6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) into cells provides a powerful tool to monitor cell migration, and to quantify cell division, because of the sequential decrease in fluorescent labeling in daughter cells. CFSE-labeled lymphocytes have been used to analyze the relationship between cell division and differentiation of cell function, and cell proliferation versus apoptosis, both in vivo and in vitro, and have allowed analysis of the site of C 2009 by John response to antigens in vivo. Curr. Protoc. Immunol. 84:4.9.1-4.9.13. Wiley & Sons, Inc. Keywords: CFSE r cell division r cell tracking r lymphocyte migration r lymphocyte positioning
INTRODUCTION The stable incorporation of the intracellular fluorescent dye 5-(and -6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) into lymphocytes (Basic Protocol) is a powerful tool to monitor lymphocyte migration in vivo, and to quantitatively analyze cell division both in vivo and in vitro (Support Protocol). The stability of CFSE labeling allows monitoring of lymphocytes over a period of months in vivo. Cell division results in sequential halving of fluorescence, and up to 8 divisions can be monitored before the fluorescence is decreased to the background fluorescence of unstained cells. The relationship between cell division and cell function is readily measured at the time of analysis by using a cell function marker (cell surface or intracellular protein) labeled with an alternate fluorochrome. Similarly, T and B lymphocyte subsets and NK cells can be individually analyzed for cell division in a complex population by using appropriate cell surface markers. CFSE remains associated with apoptotic cells for several days, and these can be analyzed together with live cells by appropriate electronic gating (by size and granularity) on the flow cytometer. Since halving of fluorescence occurs in daughter cells, by calculating the proportion of cells in each division peak and dividing by the expected progeny at those divisions (2, 4, 8, 16, etc.), the number of cells that have entered division can be calculated. This gives a precursor frequency estimate of responding cells in the cultures.
CFSE LABELING OF LYMPHOCYTES This protocol describes methods for labeling high or low numbers of lymphocytes with CFSE. Steps are provided to use CFSE-labeled cells in cell transfer studies in vivo or as cells to be cultured in vitro. If analysis of cell migration is a goal of the experiment, specific guidelines for positioning of CFSE-labeled lymphocytes in lymphoid organs or other tissues are provided in this protocol. A Support Protocol for flow cytometric analysis of CFSE-labeled cells follows.
Current Protocols in Immunology 4.9.1-4.9.13, February 2009 Published online February 2009 in Wiley Interscience (www.interscience.wiley.com). DOI: 10.1002/0471142735.im0409s84 C 2009 John Wiley & Sons, Inc. Copyright
BASIC PROTOCOL
In Vivo Assays for Lymphocyte Function
4.9.1 Supplement 84
Materials Experimental animals or human peripheral blood or cultured lymphocytes Phosphate-buffered saline (PBS; APPENDIX 2A), pH 7.4 Hanks’ balanced salt solution (HBSS), pH 7.4 (APPENDIX 2A) PBS (APPENDIX 2A) containing 5% (v/v) heat-inactivated FBS 5 mM CFSE stock solution (see recipe) Antigens and mitogens of interest 0.5 mM disodium EDTA in PBS (APPENDIX 2A) 1-ml syringes 25-G needles Fluorescence microscope with filters for fluorescein Razor blade Additional reagents and equipment for removal of mouse lymphoid organs (UNIT 1.9), preparation of mononuclear cell suspensions (UNIT 3.1), and isolation of peripheral blood mononuclear cells (UNIT 7.1), immunohistochemistry (UNIT 21.4), culturing mouse (UNITS 3.10 & 3.12), or human (UNITS 7.10 & 7.11) lymphocytes, and counting cells using a hemacytometer (APPENDIX 3A) Label lymphocytes with CFSE 1a. For high cell numbers: Prepare lymphocytes using the techniques described in UNIT 1.9 (for mice; removal of lymphoid organs), UNIT 3.1 (preparation of cell suspensions), and UNIT 7.1 (preparation of PBMC), at a concentration of 50 × 106 cells/ml in either PBS (without serum) for human PBMC or HBSS (without serum) for mouse lymphocytes. 1b. For low cell numbers: Resuspend freshly isolated lymphocytes in PBS containing 5% FBS at concentrations from 0.5 × 106 cells/ml to 10 × 106 cells/ml. At low cell concentrations it is absolutely essential that there be protein present to buffer the toxic effect of CFSE. Cultured lymphocytes that are quiescent at the end of primary culture are labeled directly in their culture medium (containing 10% FBS) after equilibrating to room temperature.
2. Dilute the stock 5 mM CFSE solution 1/100 in PBS (to give a 50 μM solution). Add 110 μl of this solution per milliliter of cells (to give a final concentration of 5 μM), and mix rapidly. After 5 min at room temperature add 10 vol of PBS containing 5% FBS, centrifuge cells 5 min at 300 × g, 20◦ C, and remove the supernatant. Wash three times, each time by resuspending in 10 vol PBS containing 5% FBS, centrifuging 5 min at 300 × g, 20◦ C, and removing the supernatant. Labeling with CFSE occurs rapidly, and it is essential that CFSE be dispersed as evenly and quickly as possible so that cells are uniformly labeled. One strategy to achieve this is to add the cell suspension into the bottom of a 10-ml plastic tube, then, while holding the tube almost horizontally, add the CFSE solution to a nonwetted portion of the plastic at the top of the tube. The tube is then capped while still in the near-horizontal position, and then rapidly inverted several times to mix the lymphocytes and CFSE solution. An alternate strategy is to predilute the CFSE to 10 μM and add an equal volume to the cell suspension while vortexing. If this strategy is used for high cell numbers, prepare the lymphocytes at 100 × 106 cells/ml instead of 50 × 106 cells/ml.
Use of CFSE to Monitor Lymphocyte Migration and Proliferation
When labeling cultured lymphocytes it is best to add CFSE directly into the existing culture medium without prior centrifugation. When cultured cells are centrifuged they form small aggregates such that individual cells are not exposed equally to CFSE. After labeling cultured lymphocytes with CFSE, the cells are washed in PBS and then incubated for 5 min in 0.5 mM EDTA in PBS to dissociate any aggregates, and washed once more in PBS before resuspending in culture medium for restimulation in culture.
4.9.2 Supplement 84
Current Protocols in Immunology
CFSE staining of lymphocytes cannot be measured directly after labeling because of the extremely high fluorescence. The majority of CFSE initially taken up by the cells is not stably incorporated and is lost within the first 24 hr after labeling.
Perform in vivo transfer of CFSE-labeled lymphocytes 3. Resuspend CFSE-labeled lymphocytes in tissue culture medium lacking added protein (no serum), and, using a 1-ml syringe equipped with a 25-G needle, inject intravenously (i.v.) into recipient animals. In the case of mice, inject into the lateral tail vein, with 1 × 106 to 40 × 106 cells being injected into each recipient mouse in a volume of 0.1 to 0.2 ml. There is a linear increase in the number of CFSE-labeled cells entering mouse lymphoid organs with the transfer of up to 50 × 106 cells, but when greater numbers of cells are transferred the system appears to become saturated. If extensive proliferation of the CFSE-labeled population is anticipated, it is essential that the cells have an independent marker for detection from host cells that does not decrease with cell division. This is most commonly satisfied by using cells that have a CD45 allotypic difference from host animals, e.g., injection of CFSE-labeled CD45.1+ cells into CD45.2+ host animals (see Critical Parameters and Fig. 4.9.1). If, on the other hand, in vivo migration of lymphocytes is being investigated under conditions of minimal cell division, it is possible to independently track two different lymphocyte populations in the same animal by labeling the cells to different fluorescence intensities with CFSE (Lyons, 1999). One population is labeled with 5 μM CFSE (see step 1a or 1b above) and the other population with one-quarter (1.25 μM) or one-sixteenth (0.312 μM) the normal CFSE-labeling concentration. If one plans to examine the CFSE-labeled cells
