Cell Tracking with Lipophilic Membrane Dyes: Applications in Cancer Research

Supplemental Information

Katharine A. Muirhead, Joseph D. Tario, Jr., Paul K. Wallace

Table S1. Non-Perturbing Membrane-Dye Labeling Conditions for Selected Cell Types1

Cell Type Final Cell Concentration Final Dye Concentration
hPBMC* (large cell number) 1 x 107 cells/mL
3 x 107 cells/mL
5 x 107 cells/mL
2 µM PKH67
4 µM CellVue® Claret
5 µM CellVue Claret
hPBMC* (low cell number) 5 x 106 cells/mL
1 x 106 cells/mL
2 µM PKH26
1 µM CellVue Claret
Cultured cell lines 1 x 107 cells/mL
1 x 107 cells/mL
1 x 107 cells/mL
1 x 107 cells/mL
1 x 107 cells/mL
15 µM PKH26 (U937)
12.5-15 µM PKH26 (U937)
1 µM PKH67 (K562)
1 µM PKH67 (polyclonal T cell lines)
10 µM CellVue Claret (YAC-1)
* A low speed wash (300 x g) post-Ficoll/Hypaque was used to minimize platelet contamination

1 Adapted from Table 1 of Tario Jr. et al. (2011); Reprinted with permission from Humana Press. First published in Methods in Mol. Biology, 699, 119 (2011).

Table S2. Choosing a Membrane Dye Kit for Cell Tracking and Proliferation Monitoring1

Dye PKH67 PKH26 CellVue® Claret
Emission GREEN (max. 502nm) ORANGE-RED (max. 567nm) FAR-RED (max. 675 nm)
Useful laser lines 457nm, 488nm 488nm, 514nm, 543nm 633-635nm, 647nm
Spectrally compatible with2 Hoechst 33342, PE, Cy 3, TR, PI, some red FPs, PE-Cy5, PerCP, 7-AAD, APC, TO-PRO-3, DRAQ5, PE-Cy7, APC-Cy7 Hoechst 33342, FITC, CFSE, green FPs, TR, PI, some red FPs, PE-Cy5, 7-AAD, APC, TO-PRO-3, DRAQ5, PE-Cy7, APC-Cy7 Hoechst 33342, FITC, CFSE, green/yellow FPs, PE, Cy 3, PE-Cy5, PI, TR, some far-red FPs, PerCP, 7-AAD, PE-Cy7, APC-Cy7
NOT suitable for use with2 FITC, CFSE, green/yellow FPs PE, Cy3, TR, PerCP, some yellow/red FPs3 some red/far-red FPs,3 APC, Cy5, DRAQ5, TO-PRO-3
Dye transfer in co-culture Minimal4 Minimal Minimal
Time to 50% intensity in non-dividing cells in vivo 10-12 days 100-200 days Not yet known (in vitro studies suggest it will be similar to PKH26)
General cell tracking applications (typical timeframe) In vitro and in vivo5 (days to weeks) In vitro and in vivo5 (days to months) In vitro6 and in vivo
(maximum not yet known)
Cell proliferation monitoring Yes5 (8-10 generations) Yes5
(8-10 generations)
Comparable to standard tracking dyes (e.g. PKH26 and CFSE)7
Sigma® Cell Linker Kit configurations PKH67GL-1KTL8
Abbreviations: APC = allophycocyanin; 7-AAD= 7-aminoactinomycin D; CFSE = carboxyfluorescein succinimide ester; Cy3 = cyanine 3; Cy5 = cyanine 5; Cy7 = cyanine 7; FPs = fluorescent proteins; PE = phycoerythrin; PI = propidium iodide; PerCP = peridinin chlorophyll lprotein; TR = Texas Red*
* CellVue is a trademark of PTI Research, Inc. CY is a trademark of GE Healthcare. Texas Red and TO-PRO-3 are trademarks of Molecular Probes. DRAQ5 is a trademark of Biostatus Ltd.

1 See Refs. 1-4 and11 for further details. Originally developed by Paul Karl Horan and colleagues at Zynaxis Cell Science in the late 1980’s, PKH26 been distributed by Sigma-Aldrich since 1993 and PKH67 since 1997. CellVue Claret, a far red analog developed by PTI Research, Inc. was added to the family of Sigma cell tracking kits in 2008.
2 Spectral compatibilities and incompatibilities are representative, not exhaustive, and assume that laser excitation spots are spatially separated, not coincident. Relative signal from different fluorescent probes will vary depending on the biological system of interest. Each user must run the necessary controls to verify compatibility in their own system.
3 Shcherbo D, Merzylak EM, Chepurnykh TV et al. Bright Far-red Fluorescent Protein for Whole-body Imaging. Nature Meth. 4:741-746 (2007).
4 <0.3% @ 24h when FBS or other protein is used as “stop” reagent in staining protocol (see text).
5 For partial bibliography of PKH dye applications (1988 – 2006), see Tables 1 and 2 at www.sigmaaldrich.com/insite_pkh_fluorescent_cell.
6 See Refs. 3, 8, 9, 12 and 20.
7 See Refs. 3 and 11.
8 GL kits contain 0.5 mL dye + 6x10mL Diluent C (recommended for general membrane labeling of cells for large or in vivo studies).
9 MIDI kits contain 2x0.1 mL dye + 6x10mL Diluent C (recommended for general membrane labeling of cells for in vitro proliferation or cytotoxicity studies).
10 MINI kits contain 0.1 mL dye + 1x10mL Diluent C (recommended for general membrane labeling of cells for small or preliminary in vitro studies).
11 PCL kits contain 0.5 mL dye + 6x10mL Diluent B (recommended for selective labeling of phagocytes in the presence of non-phagocytes for in vitro or in vivo studies).

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Staining Mechanism
Figure S1. Staining mechanism (adapted from Ref. 2)
The PKH and CellVue dyes are lipid-like molecules with fluorescent "head groups" and long aliphatic "tails" (not shown to scale). In salt-containing buffers or media they rapidly form micelles or aggregates with poor cell labeling efficiency for non-phagocytic cells. Diluent C (the iso-osmotic, salt- and solvent-free staining vehicle provided in Sigma general membrane labeling kits) maximizes dye solubility and facilitates near-instantaneous partitioning into the lipid bilayer. Strong non-covalent interactions with surrounding lipid tails provide long term dye retention and stable fluorescence intensities in non-dividing cells.
Reprinted with permission from John Wiley & Sons, Inc. First published in Cytometry, 73A. 1019 (2008).
General Membrane Labeling Protocol for PKH26, PKH67 and CellVue® Claret
Figure S2. General membrane labeling protocol for PKH26, PKH67 and CellVue Claret
When using the salt-free Diluent C vehicle provided with Sigma cell linker kits to maximize staining efficiency and homogeneity, partitioning of these highly lipophilic dyes into cell membranes occurs almost instantly upon mixing with cells. As illustrated in this schematic for labeling with PKH67, bright, uniform and reproducible staining is most readily obtained by: 1) minimizing the amount of salts present in the staining step and 2) insuring rapid homogeneous dispersal of cells in dye. (For detailed methods and staining protocols, see Refs. 2 and 3 and links to individual product bulletins included in Table S2.)
Color coding of target populations allows simultaneous comparison of in vivo CTL cytotoxicity against multiple epitopes in a single mouse
Figure S3. Color coding of target populations allows simultaneous comparison of in vivo CTL cytotoxicity against multiple epitopes in a single mouse
Unpulsed splenocytes or splenocytes pulsed with 1 of 4 immunogenic MHV-68 peptides for 1 h @ 37 °C. were labeled with distinct 1 or 2-color combinations of 3 different cell tracking dyes. Single staining with CMTMR identified unpulsed splenocytes, whereas peptide-pulsed splenocytes were labeled with combinations of CellVue Claret and CFSE. The 5 labeled target populations were then admixed in equal numbers, injected into naïve mice or mice that had been infected with MHV-68 virus 10 days prior (3 per group), and spleens were harvested 4 hours post-injection. After red cell lysis, the frequency of viable targets in each population was assessed by flow cytometry, using light scatter gating to exclude debris and aggregates and 7-AAD uptake to exclude non-viable cells. [NOTE: Although the peptides shown here were candidate epitopes for an antiviral vaccine, a similar strategy could be applied to identification of efficacious anti-tumor vaccine components.]
A. Representative two color plots showing tracking dye combinations used to identify target populations pulsed with each of the peptides (CMTMR only: No peptide; CFSEhi CellVue Claretneg: ORF61p; CFSElo CellVue Claretneg: ORF6p; CFSEneg CellVue Claretpos: ORF9p; CFSEhi CellVue Claretpos: B8Rp).
B. Representative single color histograms used to determine epitope specificity of killing. Left panels: CMTMR gated. Center panels: CFSE distribution of CellVue Claretneg targets. Right panels: CFSE distribution of CellVue Claretpos targets). Values on lower panels indicate percent specific lysis, calculated based on the change in frequency of viable targets seen in infected vs. naïve animals (see Ref. 9 for details).
Reprinted with permission from Informa Healthcare. First published in Immunol. Invest.36, 829 (2007).

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