Lipophilic Dye Kits for Cell Tracking

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
Cell Linker Kit configurations	PKH67GL-1KTL8 MIDI67-1KT9 MINI67-1KT10	PKH26GL-1KT8 MINI26-1KT10 PKH26PCL-1KT11	MIDCLARET-1KT9 MINCLARET-1KT10

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 Cytiva. 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 us since 1993 and PKH67 since 1997. CellVue Claret, a far red analog developed by PTI Research, Inc. was added to our family of 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.
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).

Figure S1. Staining mechanism

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 our 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).

Figure S2. General membrane labeling protocol for PKH26, PKH67 and CellVue Claret

When using the salt-free Diluent C vehicle, provided with our 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.)

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; CFSE^hi CellVue Claret^neg: ORF61p; CFSE^lo CellVue Claret^neg: ORF6p; CFSE^neg CellVue Claret^pos: ORF9p; CFSE^hi CellVue Claret^pos: 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 Claret^pos 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).

References

1. Schwaab T, Fisher JL, Meehan KR, Fadul CE, Givan AL, Ernstoff MS. 2007. Dye Dilution Proliferation Assay: Application of the DDPA to Identify Tumor-Specific T Cell Precursor Frequencies in Clinical Trials. Immunological Investigations. 36(5-6):649-664. https://doi.org/10.1080/08820130701674760

2. Wallace PK, Tario JD, Fisher JL, Wallace SS, Ernstoff MS, Muirhead KA. 2008. Tracking antigen-driven responses by flow cytometry: Monitoring proliferation by dye dilution. Cytometry. 73A(11):1019-1034. https://doi.org/10.1002/cyto.a.20619

3. Tario JD, Muirhead KA, Pan D, Munson ME, Wallace PK. 2011. Tracking Immune Cell Proliferation and Cytotoxic Potential Using Flow Cytometry.119-164. https://doi.org/10.1007/978-1-61737-950-5_7

4. Poon RYM, Ohlsson-Wilhelm BM, Bagwell CB, Muirhead KA. 2000. Use of PKH Membrane Intercalating Dyes to Monitor Cell Trafficking and Function.302-352. https://doi.org/10.1007/978-3-642-57049-0_26

5. Zaritskaya L, Shurin MR, Sayers TJ, Malyguine AM. 2010. New flow cytometric assays for monitoring cell-mediated cytotoxicity. Expert Review of Vaccines. 9(6):601-616. https://doi.org/10.1586/erv.10.49

6. Gertner-Dardenne J, Poupot M, Gray B, Fournié J. 2007. Lipophilic Fluorochrome Trackers of Membrane Transfers between Immune Cells. LIMM. 36(5):665-685. https://doi.org/10.1080/08820130701674646

7. Daubeuf S, Aucher A, Bordier C, Salles A, Serre L, Gaibelet G, Faye J, Favre G, Joly E, Hudrisier D. Preferential Transfer of Certain Plasma Membrane Proteins onto T and B Cells by Trogocytosis. PLoS ONE. 5(1):e8716. https://doi.org/10.1371/journal.pone.0008716

8. HoWangYin K, Alegre E, Daouya M, Favier B, Carosella ED, LeMaoult J. 2010. Different functional outcomes of intercellular membrane transfers to monocytes and T cells. Cell. Mol. Life Sci.. 67(7):1133-1145. https://doi.org/10.1007/s00018-009-0239-4

9. Bantly AD, Gray BD, Breslin E, Weinstein EG, Muirhead KA, Ohlsson-Wilhelm BM, Moore JS. 2007. CellVue® Claret, a New Far-Red Dye, Facilitates Polychromatic Assessment of Immune Cell Proliferation. Immunological Investigations. 36(5-6):581-605. https://doi.org/10.1080/08820130701712461

10. Lipscomb MW, Taylor JL, Goldbach CJ, Watkins SC, Wesa AK, Storkus WJ. 2010. DC expressing transgene Foxp3 are regulatory APC. Eur. J. Immunol.. 40(2):480-493. https://doi.org/10.1002/eji.200939667

11. Barth RJ, Fisher DA, Wallace PK, Channon JY, Noelle RJ, Gui J, Ernstoff MS. 2010. A Randomized Trial of Ex vivo CD40L Activation of a Dendritic Cell Vaccine in Colorectal Cancer Patients: Tumor-Specific Immune Responses Are Associated with Improved Survival. Clinical Cancer Research. 16(22):5548-5556. https://doi.org/10.1158/1078-0432.ccr-10-2138

12. Morse MD, McNeel DG. 2010. Prostate cancer patients on androgen deprivation therapy develop persistent changes in adaptive immune responses. Human Immunology. 71(5):496-504. https://doi.org/10.1016/j.humimm.2010.02.007

13. Basu D, Nguyen TK, Montone KT, Zhang G, Wang L, Diehl JA, Rustgi AK, Lee JT, Weinstein GS, Herlyn M. 2010. Evidence for mesenchymal-like sub-populations within squamous cell carcinomas possessing chemoresistance and phenotypic plasticity. Oncogene. 29(29):4170-4182. https://doi.org/10.1038/onc.2010.170

14. Schubert M, Herbert N, Taubert I, Ran D, Singh R, Eckstein V, Vitacolonna M, Ho AD, Zöller M. 2011. Differential survival of AML subpopulations in NOD/SCID mice. Experimental Hematology. 39(2):250-263.e4. https://doi.org/10.1016/j.exphem.2010.10.010

15. Kusumbe AP, Bapat SA. 2009. Cancer Stem Cells and Aneuploid Populations within Developing Tumors Are the Major Determinants of Tumor Dormancy. Cancer Research. 69(24):9245-9253. https://doi.org/10.1158/0008-5472.can-09-2802

16. Cicalese A, Bonizzi G, Pasi CE, Faretta M, Ronzoni S, Giulini B, Brisken C, Minucci S, Di Fiore PP, Pelicci PG. 2009. The Tumor Suppressor p53 Regulates Polarity of Self-Renewing Divisions in Mammary Stem Cells. Cell. 138(6):1083-1095. https://doi.org/10.1016/j.cell.2009.06.048

17. Pece S, Tosoni D, Confalonieri S, Mazzarol G, Vecchi M, Ronzoni S, Bernard L, Viale G, Pelicci PG, Di Fiore PP. 2010. Biological and Molecular Heterogeneity of Breast Cancers Correlates with Their Cancer Stem Cell Content. Cell. 140(1):62-73. https://doi.org/10.1016/j.cell.2009.12.007

18. Gordon EJ, Rao S, Pollard JW, Nutt SL, Lang RA, Harvey NL. 2010. Macrophages define dermal lymphatic vessel calibre during development by regulating lymphatic endothelial cell proliferation. Development. 137(22):3899-3910. https://doi.org/10.1242/dev.050021

19. Engels N, König LM, Heemann C, Lutz J, Tsubata T, Griep S, Schrader V, Wienands J. 2009. Recruitment of the cytoplasmic adaptor Grb2 to surface IgG and IgE provides antigen receptor?intrinsic costimulation to class-switched B cells. Nat Immunol. 10(9):1018-1025. https://doi.org/10.1038/ni.1764

20. Ring S, Karakhanova S, Johnson T, Enk AH, Mahnke K. 2010. Gap junctions between regulatory T cells and dendritic cells prevent sensitization of CD8+ T cells. Journal of Allergy and Clinical Immunology. 125(1):237-246.e7. https://doi.org/10.1016/j.jaci.2009.10.025

21. Megjugorac NJ, Jacobs ES, Izaguirre AG, George TC, Gupta G, Fitzgerald-Bocarsly P. 2007. Image-Based Study of Interferongenic Interactions between Plasmacytoid Dendritic Cells and HSV-Infected Monocyte-Derived Dendritic Cells. Immunological Investigations. 36(5-6):739-761. https://doi.org/10.1080/08820130701715845