Cell division tracking using CFSE Bruce Lyons, Division of Haematology, IMVS, Hanson Institute, Adelaide

Workshop outline: - Introduction to CFSE. - Some of the crucial things you need to consider. - A basic protocol around which to plan your application. -Coffee break? - Question time: *specific problems you may have been experiencing. *discussion about any applications you are considering. *helping other investigators at your institute. Bruce.Lyons@imvs.sa.gov.au (08) 8222 3590

The original observation Lyons (1999) Immunol. Cell Biol. 77, 509

Flow Cytometric uses of CFSE CFSE or CFDA-SE: carboxyfluorescein diacetate, succinimidyl ester. A fluorescein derivative which is cell permeant and non-fluorescent; cellular esterases cleave acetate groups, rendering the molecule fluorescent and cell impermeant. Succinimidyl ester binds to free amines, resulting in long lived fluorescent adducts.

CFSE Structure O O O CH3-C-O O-C-CH3 O O O N-O-C O O

Applications CFSE is partitioned equally among daughter cells with each division. Useful for both in vitro studies, and analysis of cell behavior when transferred in vivo. CFSE has very bright fluorescence, therefore location of cells in sections can be determined using confocal or fluorescence microscopy. These properties allows simultaneous analysis of cell number, position, as well as division status. Fluorochromes compatible with fluorescein can be used to probe other cellular properties.

Dilution of CFSE with cell division Divisions: 3 2 1 0 Divisions: 3 2 1 0 Lyons and Parish, 1994 JIM, 171;131 CFSE FL-1 (Log)

Properties of CFSE The fluorescence intensity per cell obtained is proportional to CFSE concentration, and duration of staining. This allows differential staining of populations of cells. Even at six months, undivided cells retain about 10% of their original fluorescence. This makes the dye useful for long term in vivo tracking experiments. Viable cells from defined division cycles can be sorted for further analysis.

CFSE Fluorescence intensity is proportional to dye concentration at the time of staining

Using differential labelling to track survival of different cell populations Quarter labelled Fully labelled Fulcher et al (1996) J. Exp. Med. 183; 2313

CFSE intensity drops rapidly in the first few days in the absence of cell division, but then stabilises

Cell division profiles are not affected by rapid CFSE loss in the first few days after staining- this loss of stain due to catabolism is proportional. Lee, Hodgkin and Lyons (1996), J.Exp.Med. 184; 277

Measurement of cell properties simultaneously with CFSE Surface molecule expression, immunophenotype DNA content, 7-AAD stain after fixation Incorporation of BrdU into DNA Apoptosis, Annexin V + 7-AAD Cytokine expression, fix/permeabilise/stain Nuclear preparations, transcription factor levels Modeling kinetics of cell division Analysis of asynchronously dividing cells

Division tracking of cell nuclei to examine transcription factor translocation to the nucleus (Hasbold & Hodgkin 2000)

Maximising resolution Nordon et al 1997- pre-sorted heterogeneously staining cells over a narrow range of CFSE fluorescence to improve peak resolution. Need to ensure this does not select on the basis of size etc. and bias results. 40 channel interval CFSE

progenitor proliferation Using CFSE to track CML progenitor proliferation SDM SDM + Glivec

CFSE division tracking allows the identification of the division in which cell death occurred: Division number: 3 2 1 0 L: 4 20 34 44 D: 1 6 22 71 Live: Dead:

Calculating precursor frequency 0 1590 /1 1590 1 1140 /2 570 2 1388 /4 347 3 687 /8 86 4 41 /16 2.6 Total; 2595 Cohort Total; 1005.6 % original cells dividing is 1005.6divided by (2595 + 1005.6) = 38.7% Lyons (2000) J. Imm. Meth. 243, 147

Courtesy P Viboonjuntra CML CD34 cells: Colchicine stops division and allows gating strategy to be set up FS Gate on R1 SS FS R1 CFSE CD34 PE Ungated Courtesy P Viboonjuntra CFSE

CML CD34 cells stimulated with GM-CSF at 300 pg/ml FS SS FS Gate on R1 R1 CFSE CD34 PE Ungated Courtesy P Viboonjuntra CFSE

CML CD34 cells stimulated with GM-CSF Patient 2 Patient 1 CD34 PE CD34 PE 7-AAD Courtesy P Viboonjuntra 7-AAD CFSE

Calculation of Proliferative Index (P.I.) GATE GEN. EV. f #Parent R 3 0 561 1 561 R 4 1 1158 2 579 R 5 2 2311 4 578 R 6 3 1541 8 193 R 7 4 142 16 9 SUM: 5713 1920 P.I. 5713/1920 = 2.98 4 3 2 1 0 CFSE ---> The Proliferative Index is the sum of the cells in all generations divided by the calculated number of original parent cells. It is useful for comparing quantity of cell division between cultures of the same cells undergoing different treatments.

Using proliferative index to compare response to cytokines and anti-proliferative effects of drugs GM-CSF Courtesy P Viboonjuntra

Analysis of rare populations of lymphocytes (< 2%) after in vivo transfer- gating on CFSE positive events to exclude host cells Lyons (1997) Immunol. Cell Biol. 75; 7

Examining cell division linked differentiation CD44 IgD IgM IgG1 Lee, Hodgkin and Lyons (1996), J.Exp.Med. 184; 277

Compensation Substantial overlap of Fluorescein emission into PE channel makes compensation of CFSE challenging! Baumgarth and Roederer (2000) J Immunol. Meth. 243; 77

Compensation can be very tricky when CFSE is bright ! No PE PE- anti-B cell Uncompensated Compensated 0.5% Fl-2 - Fl-1 52% Fl-1 - Fl-2

Fl-1 gain 590 Fl-1 gain 520 Fl-1 gain 620 under-compensated over-compensated -> If you are having trouble compensating, dropping the Fl-1 gain will make matters worse! -> Decreasing the Fl-2 gain can allow very bright CFSE to be compensated -> Can set Fl-1 - Fl2 to about 50-60%, and then use gain controls to ensure accurate compensation

A brief protocol for CFSE staining and analysis Cells: Some cells are more suited to CFSE analysis than others. Small resting lymphocytes are most suitable. Activated lymphocytes are susceptible to apoptosis after labelling. Cell lines give very broad levels of staining making resolution of divisions difficult. Haemopoietic progenitors are fairly heterogeneous, but pre-sorting a 40 channel population can overcome this.

Level of CFSE staining: This will need to be determined empirically for each type of experiment. Need to consider effects on cell viability and ability to compensate when CFSE intensities are high. For experiments examining division up to a week, 10 micromol CFSE for 10 min at 37 degrees is a good starting point. For long term tracking in vivo higher levels of around 50 micromolar should be used.

Staining Cells: Duration of analysis - < 7 days : stain with 5-10 micromol CFSE for 10 min in PBS/0.1%BSA, with cells at 5x107/ml or less. > 7 days : stain with 25-50 micromol CFSE. (note that even uniform staining requires good mixing; I usually make up a 2x CFSE solution and immediately mix with a 2x cell suspension) To stop staining, add 5 volumes of ice cold medium or PBS containing protein (10% FCS or 1% BSA) and wash 3 times with the final culture or injecting medium.

Resolution: The more heterogeneous the cell size, the lower the resolution of division peaks. Pre-sorting cells based on a narrow 40 channel gate of CFSE intensity can be used to get enhanced resolution. Control for undivided cells: You need to know the undivided peak fluorescence in order to analyse data properly; e.g. for calculating precursor percentage. In some cases, division can be stopped by colchicine. Often, there will be a proportion of non-dividing cells either in culture or in vivo, acting as an internal control.

Culture cells or inject into recipients: Make sure you have a control for determining the undivided peak! This must be incubated for the same duration as the labelled cells! Also, it is crucial to have some non-CFSE labelled cells to determine the autofluorescence level of divided cells, as this will determine the number of divisions resolvable. Analysis: Harvest cells from culture or organs of interest. Stain with antibodies or other probes.

Set up the flow cytometer: Initially, it is helpful to start off with a standard two colour set up. Adjust Fl-1 and Fl-2 gain so unlabelled resting cells sit just off axis within the first decade on a log scale, and undivided CFSE labelled cells are still on scale. Compensate for CFSE bleeding into Fl-2 channel. If you require more than 60-80% for this, you may need to drop your initial staining levels. Alternatively, try manipulating the Fl-1 and Fl-2 gains; either increase Fl-1 gain or decrease Fl-2 gain. Compensate for Fl-2 bleeding into Fl-1; this will usually be < 0.5%

Collecting events: Once the cytometer is set up for the first timepoint, the same settings should be used for the duration of the experiment. Collect files of control CFSE labelled undivided cell events as well as control non-CFSE labelled cells for each timepoint. For adoptive transfer experiments, collect a file of total events to determine percentages, then a gated set of CFSE positive events for detailed analysis of transferred cells.

Analysing the data: Interval analysis. The most basic way to analyse data is to set markers over peaks on histograms using the mean undivided cell CFSE fluorescence, minus autoflourescence, as a starting point. Boundaries are +/- 0.15log10 either side of each cell division peak. Using computer programs to deconvolute data. Sometimes, cell heterogeneity results in obscured peaks. In such cases, ModFit (Verity Software) or other programs can be very useful for obtaining accurate numbers of cells in each division cycle.

Alternatives to CFSE PKH26 This is a membrane inserting dye which is excited at 488nm and has red/orange fluorescence. Reports suggest that there can be bystander labelling of cells, and resolution is lower than CFSE. SNARF-1 carboxylic acid acetate, succinimidyl ester Developed by Molecular probes as an orange/red emitting alternative to CFSE. It has less intense fluorescence, and it may be more pH dependent than CFSE, and thus activation status sensitive.

Copies of these review articles will be provided for workshop participants: Lyons and Doherty (1998) Flow cytometric analysis of cell division. Current protocols in Cytometry Unit 9.11 - a practically oriented protocol with a troubleshooting section. Lyons, Hasbold and Hodgkin (2001) Flow cytometric analysis of cell division history using dilution of CFSE, a stably integrated fluorescent probe. Methods in Cell Biology 63; 375 - an extensive review of the applications of CFSE division analysis.

Useful References: Lyons, A.B. and Parish, C.R. 1994. Determination of lymphocyte division by flow cytometry. J. Immunol. Methods 171:131-137. Nordon, R.E., Ginsberg, S.S., and Eaves, C.J. 1997. High-resolution cell division tracking demonstrates the Flt3-ligand-dependence of human marrow CD34+CD38- cell production in vitro. Br. J. Haematol. 98:528-539. Lyons, A.B. 1999. Divided we stand: Tracking cell proliferation with carboxyfluorescein diacetate succinimidyl ester. Immunol. Cell Biol. 77:509-515. (plus eight thematically related articles) Lyons, A.B. 2000. Analysing cell division in vivo and in vitro using flow cytometric measurement of CFSE dye dilution. J. Immunol. Meth. 243:147-154

Lyons, A.B. and Doherty, K.V. 1998. Flow cytometric analysis of cell division by dye dilution. Current Protocols in Cytometry, Unit 9.11 Hasbold, J. and Hodgkin P.D. 2000. Flow cytometric cell division tracking using nuclei. Cytometry 40:230-237. Lyons, A.B., Hasbold, J. and Hodgkin, P.D. 2001. Flow cytometric analysis of cell division history using dilution of carboxyfluoresceindiacetate succinimidyl ester, a stably integrated fluorescent probe. Methods in Cell Biol. 63:375-398. Parish, C.R. and Warren, H.S. 2001. Use of the intracellular fluorescent dye CFSE to monitor lymphocyte migration and proliferation. Current Protocols in Immunology, Unit 4.9

Acknowledgements and thanks: Pongtep Viboonjuntra and Andrea Dewar for generously providing data slides Members of the Melissa White Leukaemia Research Laboratory at the Institute for Medical and Veterinary Science. Those researchers citing the original reference! (>450 so far!)