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Use of the Intracellular Fluorescent Dye CFSE to Monitor Lymphocyte Migration and Proliferation

UNIT 4.9

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 basic 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 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.

BASIC PROTOCOL

Materials Experimental animals or human peripheral blood or cultured lymphocytes Phosphate-buffered saline( PBS; APPENDIX 2), pH 7.4 Hanks’ balanced salt solution (HBSS), pH 7.4 (APPENDIX 2) PBS (APPENDIX 2) 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 2) Fluorescence microscope with filters for fluorescein 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), and culturing mouse (UNITS 3.10 & 3.12), or human (UNITS 7.10 & 7.11) lymphocytes 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 Contributed by Christopher R. Parish and Hilary S. Warren Current Protocols in Immunology (2001) 4.9.1-4.9.10 Copyright © 2001 by John Wiley & Sons, Inc.

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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 ml 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 the 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 non-wetted 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 ìM 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. 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. 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 few days.

Perform in vivo transfer of CFSE-labeled lymphocytes 3. Resuspend CFSE-labeled lymphocytes in tissue culture medium lacking added protein (no serum) and inject intravenously (i.v.) into recipient animals. In the case of mice, inject into the lateral tail vein, with from 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 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 ìM CFSE (see step 1a or b above) and the other population with one-quarter (1.25 ìM) or one-sixteenth (0.312 ìM) the normal CFSE-labeling concentration. Use of CFSE to Monitor Lymphocyte Migration and Proliferation

If one plans to examine the CFSE-labeled cells less than 24 hr after they have been transferred into recipient animals, in order to avoid off-scale fluorescence intensities on the flow cytometer, the lymphocytes should be labeled with one-quarter (1.25 ìM) or one-eighth (0.625 ìM) the normal CFSE-labeling concentration (see step 1 or 2 above).

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Current Protocols in Immunology

4. Detect the positioning of CFSE-labeled lymphocytes within lymphoid organs and other tissues by fluorescence microscopy with filter settings for fluorescein, or by immunohistochemistry (UNIT 21.4) using fluorescein-specific antibodies (Garton and Schoenwolf, 1996; Graziano et al., 1998). For fluorescence microscopy detection of CFSE-labeled cells, remove organs from animals, cut ∼3-mm sections of the organs with a razor blade, and place the sections on microscope slides for examination. CFSE fluorescence is rapidly quenched when tissue sections are viewed by fluorescence microscopy and is totally quenched when the sections are treated with conventional histological stains. For short-term positioning studies of relatively low resolution, the DNA-intercalating dye H33342 is recommended as a highly fluorescent, quenching-resistant dye for labeling lymphocytes (Parish, 1999). If high-resolution positioning studies are required, immunohistochemical detection of CFSE-labeled cells in tissue sections is recommended.

Culture lymphocytes 5. Resuspend CFSE-labeled lymphocytes in culture medium and stimulate in vitro with antigens or mitogens of interest. Procedures for culturing mouse and human lymphocytes are detailed in UNITS 3.10 & 3.12, and UNITS 7.10 & 7.11, respectively.

Harvest cells 6a. For in vivo harvesting: Collect lymphoid organs, and make single-cell suspensions (UNITS 1.9 & 3.1). 6b. For in vitro harvesting: Harvest cells from culture, wash once in 3 ml PBS, resuspend in 2 ml 0.5 mM EDTA/PBS, and incubate for 5 min at 37°C to dissociate aggregates. Centrifuge cells at 5 min at 300 × g, 20°C, resuspend in PBS containing 5% FBS, and transfer to tubes suitable for use with the flow cytometer. If the cells are to be counted manually with a hemacytometer (APPENDIX 3A), retain a small sample on ice in a separate tube or V-well plate. Cells for analysis on the flow cytometer are not fixed with paraformaldehyde, but are kept on ice until analyzed. For multiple cultures, it is convenient to use 96-well V-well plates. For cultures harvested from 96-well flat well plates, an aliquot of supernatant from each of the wells is first discarded so that the triplicate cultures can be combined into 1 well of the 96-V-well plate. Subsequent washing and treatment with EDTA then occurs in 150-ìl volumes in these wells. The plates are centrifuged for 2 min at 300 × g in a plate holder. The supernatant is removed using a microtiter pipettor by following the meniscus down while aspirating until the tip reaches the intersection of the vertical wall and the top of the V-base of the well. The pellet is mixed by agitating the plate over a vortex mixer prior to adding the next wash solution with a multipipettor.

ANALYSIS OF CFSE-LABELED CELLS BY FLOW CYTOMETRY This protocol summarizes the steps used to analyze CFSE-labeled cells in order to quantitate cell division. The proportion of cells in the individual CFSE peaks can be determined manually or by applying software that deconvolutes the peaks. Guidelines for either approach are provided in this protocol. The Critical Parameters section of this unit describes important parameters related to the analysis of CFSE-labeled cells that have been transferred in vivo or cultured in vitro following labeling. Additional information about flow cytometric analysis of cells can be found in Chapter 5. In this protocol, the choice of which other labeling procedures are to be used (e.g., cell surface staining,

SUPPORT PROTOCOL

In Vivo Assays for Lymphocyte Function

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intracellular staining for cytokines, identification of apoptotic cells) depends on the goals of the experiment at hand. Materials CFSE-labeled cells (see Basic Protocol) Analytical flow cytometer capable of 3-color fluorescence Cell sorting flow cytometer (for some applications) Additional reagents and equipment for cell surface staining of lymphocytes (UNIT 5.3), intracellular cytokine staining (UNIT 6.24), analytical flow cytometry (UNIT 5.4), flow cytometric analysis of apoptosis (UNIT 3.17), and cell sorting flow cytometry (UNIT 5.1). 1. Perform flow cytometric staining steps suited to the experiment being performed (e.g., cell surface staining, UNIT 5.3; intracellular cytokine staining, UNIT 6.24; propidium iodide staining for measurement of apoptosis, UNIT 3.17). Perform flow cytometric analysis (UNIT 5.4). CFSE is a fluorescein-based dye. Labeling lymphocytes for cell surface staining or intracellularly for cytokines requires the use of an alternative fluorochrome such as PE or Cy5 (see Table 6.21.2).

For software-based calculations 2a. Deconvolute CFSE peaks using appropriate software. Several software programs, such as Profit for Macintosh (Quantumsoft), Peakfit for PC (SPSS Sciences), ModFit (Verity Software House; see Internet Resources), and CFSE Modeler (Science Speak; see Internet Resources) are available for deconvoluting CFSE peaks.

For manual calculations 2b. Record the geometric mean fluorescence of the control CFSE stained cells and the unstained cells of both the control and stimulated cultures. For example, one might have a geometric mean fluorescence of 600 for CFSE staining of control unstimulated cells compared to an autofluorescence control of 2 for stimulated unstained cells. Note that the autofluorescence value of the unstained cells is greater for the dividing compared to the control nondividing cells.

3b. Subtract the geometric mean fluorescence of the appropriate control from that of the CFSE control cells (for example, 600 − 2 = 598). 4b. Convert this value to its base 10 logarithm (in the above case, 2.776). 5b. Determine the geometric mean fluorescence of daughter populations. As these occur at 1/2, 1/4, etc. of the undivided peak, subtract 0.3 log10 units. Thus, continuing with the above example, the successive peaks are at 2.476 (division 1), 2.176 (division 2), 1.876 (division 3), 1.576 (division 4), 1.276 (division 5), 0.976 (division 6), and 0.676 (division 7).

6b. Determine the boundaries of the peaks.

Use of CFSE to Monitor Lymphocyte Migration and Proliferation

The boundaries are midway between successive peaks and are therefore 0.15 log10 units each side of the peak. So the lower (left) boundary for each peak is as follows: 2.626 (undivided control cells), 2.326 (division 1), 2.026 (division 2), 1.726 (division 3), 1.426 (division 4), 1.126 (division 5), 0.826 (division 6), 0.526 (division 7). Now determine the antilog of these figures: 423, 212, 106, 53.2, 26.7, 13.4, 6.7, 3.4. To these values are added the geometric mean autofluorescence (2 in this example) to give 425, 214, 108, 55.2, 28.7, 15.4, 8.7, 5.4.

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7b. Apply these markers using a flow cytometry program (such as Cell Quest, Becton Dickinson) and determine the % of cells in each peak. 8b. Determine the actual number of cells in each peak from the manual counts or counting bead analysis. 9b. Divide the cell numbers in each peak by the expected progeny for those divisions. Thus divide by 2 for 1 division, divide by 4 for 2 divisions, divide by 8 for 3 divisions, etc. The total of these numbers gives the number of progenitor cells and can be compared with the number of cells in control cultures. In some cases applying the above calculations gives markers that are not aligned exactly over the peaks. When the calculations are redone using division 1 as a reference peak, the markers then align correctly. It appears that during the transition from undivided to the first division there is some loss of incorporated CFSE. This appears to occur with some cell types more than others. The example given in Figure 4.9.1 for human peripheral blood lymphocytes responding to phytohemagglutinin is a case in point. You will note that the geometric mean for the undivided cells is 763 which is more than twice that of the peak in division 1. The remainder of the peaks are close to the expected halving with each division. This quantitative analysis of cell division giving the number of progenitor cells provides useful information. First, the lymphocyte population may be stimulated to divide and to

Figure 4.9.1 CFSE profiles for PBMC labeled at 1 × 10 6/ml in PBS containing 5% FBS with 5 µM CFSE for 5 min and cultured with 5 µM phytohemagglutinin for 4 days. The unfilled peak on the right at 7.6 × 102 fluorescence units corresponds to the control unstimulated CFSE-labeled cells. The unfilled peak on the left at 2 × 100 fluorescence units corresponds to the autofluorescence of unlabeled cells. The calculated marker positions are shown: M1 (undivided), M2 (1 division), M3 (2 divisions), M4 (3 divisions), M5 (4 divisions), M6 (5 divisions), and M7 (6 divisions).

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undergo activation-induced cell death. Dead cells retain the CFSE dye and thus the division number in which death of a cell occurred can be readily determined. Dead cells can be identified by forward and side scatter or by propidium iodide uptake. Comparison of the number of progenitor cells in the stimulated and control cultures will show if significant cell death has occurred. Second, the percentage of progenitor cells in divisions 1 through 7 compared to divisions 0 through 7 is an estimate of the precursor frequency, i.e., the proportion of the lymphocyte population with reactivity to a particular antigen or mitogen. In the experiment shown in Figure 4.9.1, 29.63% of cells have entered division. The limitation using this technique for the determination of precursor frequencies is the number of divisions that occur before the fluorescence of the dividing cells merges with the autofluorescence of unstimulated cells.

REAGENTS AND SOLUTIONS Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see APPENDIX 2A; for suppliers, see APPENDIX 5.

CFSE solution Dissolve 5- (and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes) in dimethylsulfoxide at a final concentration of 5 mM. Store in suitably sized aliquots at −20°C. The stock solution can be refrozen provided this is done as soon as possible after thawing. Prepare the substock of diluted CFSE just before use. Do not refreeze the substock.

COMMENTARY Background Information

Use of CFSE to Monitor Lymphocyte Migration and Proliferation

Unlike most other cells in vertebrates, the cells of the immune system have the remarkable ability to continually migrate throughout the body and position themselves in specific locations within tissues, particularly within lymphoid organs. In addition, following contact with antigen, there is a rapid clonal expansion of antigen-specific T and B lymphocytes, with the migration and positioning behavior of the proliferating lymphocytes often being very different from their precursors. In order to fully understand a functioning immune system, techniques are required that can simultaneously follow lymphocyte proliferation, lymphocyte migration into different lymphoid organs, and the positioning pattern of the lymphocytes within lymphoid organs. Early studies employed radioactive markers to measure lymphocyte proliferation and follow lymphocyte migration. [3H]thymidine has been widely used to follow lymphocyte proliferation, both in vitro (UNIT 7.10) and in vivo. However, this approach suffers from the disadvantage that it is difficult to assess the subpopulation of lymphocytes that is proliferating, tedious autoradiography procedures being required to enumerate the proliferating cells. Furthermore, the procedure only measures those cells that are in S phase at the time of [3H]thymidine addition. Bromodeoxyuridine

(UNIT 4.7) is an excellent reagent for measuring lymphocyte turnover in vivo but yields limited migration and positioning information. The γemitting isotope 51Cr has been used for many years to follow the distribution pattern of injected lymphocytes in vivo but has a number of technical limitations (Ford, 1978), and yields no information about the phenotype, proliferation status and positioning pattern of the injected cells. During the last 20 years, at least 14 different fluorescent dyes have been used to monitor lymphocyte migration (Parish, 1999), with the fluorescein-based dye CFSE emerging as the most versatile fluorescent dye. CFSE is a membrane-permeant dye that can very stably label cells by covalently coupling to intracellular molecules. CFSE...