Vybrant™ DiO, DiI and DiD Dyes for Multiple Labeling of Red Blood Cell Populations for In Vivo Survival Studies.
Authors: Armstrong Jonathan K. , Wenby Rosalinda B. , Meiselman Herbert J. , Fisher Timothy C. 
Published: October 2019

Abstract
Background: Studies of the circulatory time of red blood cells (RBCs) or drug delivery vehicles such as liposomes require a radiolabel (e.g., 51Cr), a fluorescent probe or an affinity tag (e.g., biotin). These techniques may be complicated by progressive loss of the probe from the labeled cells, transfer to other cells, and sometimes by the development of an immune response to the label. Long chain dialkylcarbocyanines (Vybrant DiO, DiI and DiD) are bright, stable lipophilic membrane fluorescent labels that can exhibit fluorescence resonance energy transfer (FRET) and are easily observed by flow cytometry using standard filters (FL1, FL2 and FL4 detection channels respectively). The staining is very stable in vivo and is dose-dependent, thus populations of labeled cells may have discrete fluorescence intensities. We recognized that use of these dyes in combination could permit the simultaneous observation of several different populations of cells within the same subject, together with one or more internal controls. To evaluate this technique, we developed procedures suitable for the fluorescent labeling up to 14 discrete populations of human or rabbit RBCs. The properties of the rabbit RBCs were then evaluated in vivo.
Methods: Washed human RBCs were suspended at a 10% hematocrit in 20mM HEPES buffer, and added to an equal volume of freshly prepared 1, 4 or 8 μM DiO, DiI, DiD, or mixtures of two labels; 36 different combinations in total. The cells were incubated with the dye for 60 minutes at 37°C, washed twice in HEPES with 0.1% albumin and examined by flow cytometry. Aliquots from 6 to 15 differently-labeled samples were then combined, and the mixtures re-examined. For in vivo studies, the labeling procedure was repeated with autologous rabbit RBCs at dye concentrations of 1.5, 6, and 12 μM. The labeled rabbit RBCs were washed, suspended in saline and reinfused via the ear vein. Small blood samples were drawn daily for 4 days and weekly for 5 weeks, and the number of fluorescently labeled cells of each color remaining was determined by flow cytometry.
Results: Of the 36 possible combinations, 14 discrete populations could be clearly distinguished from “dot-plots” of FL1 vs. FL2, FL1 vs. FL4 and FL2 vs. FL4. FRET was observed for all cells stained with two labels. In vivo evaluation of 8 different labeled rabbit RBC populations showed normal survival. A 10–20% reduction in fluorescence intensity was observed over the study period; this reduction did not compromise the clear identification of each discrete population. Rabbits that were repeatedly exposed to Vybrant-labeled autologous RBCs (up to 15 exposures over 3 years) showed no evidence of accelerated clearance, suggesting that these fluorescent labels do not exhibit significant immunogenicity.
Conclusion: The use of two long-chain dialkylcarbocyanines in combination is ideal for studies which require multiple cell populations to be followed within the same experiment, and additionally allows for one or more internal controls to correct for any variability of circulation times. The technique is presently being used for studies of RBC senescence in which multiple discrete density populations of RBCs are individually labeled. If these dyes can be shown to have an acceptably low toxicity, the ability to track multiple populations could also be useful for some clinical applications, e.g., to simultaneously evaluate the survival of RBCs from many different donors in patients with multiple alloantibodies, to identify compatible units for transfusion.