Protocol update for late endothelial progenitor cells identified by double‐positive staining

Abstract Endothelial progenitor cells (EPCs), which are precursors of endothelial cells (ECs), have the capacity to circulate, proliferate and differentiate into mature ECs. EPCs are primarily identified by the uptake of 1,1‐dioctadecyl‐3,3,3,3‐tetramethylindocarbocyanine‐labelled acetylated low‐density lipoprotein (Dil‐acLDL) and the binding of fluorescein‐isothiocyanate (FITC)‐conjugated Ulex europaeus agglutinin lectin (FITC‐UEA‐I). However, the cytoplasm and nucleus are usually stained by FITC‐UEA‐I via a typical method to double‐stain late EPCs. It is necessary to explore a new method to improve the quality of fluorescence photomicrographs of late EPCs stained with FITC‐UEA‐I. Here, we described an updated protocol for double‐staining late EPCs with Dil‐acLDL and FITC‐UEA‐I, with the cells more optimally stained with FITC‐UEA‐I.

(FITC-UEA-I), as determined by fluorescence microscopy [7][8][9][10][11] ; these cells were further shown to express endothelial cell (EC) markers such as von Willebrand factor (vWF), kinase insert domain receptor (KDR), CD105, CD146 and CD144 but not haematopoietic cell-specific surface antigens, including CD11b, CD14 and CD45. 12 Fluorescent staining was used to detect the uptake of DiI-acLDL and the binding of FITC-UEA-I. Typically, the protocol for double-staining EPCs [8][9][10][11] was as follows: Adherent cells were first incubated with DiI-acLDL for 1-3 hours and were then incubated with FITC-UEA-I for 1 hour after being fixed for 10 min. Although the protocol was not complicated (Figure 1(left)), it is not easy to obtain good-quality fluorescence photomicrographs of the binding of FITC-UEA-I to EPCs with an inverted fluorescence microscope. FITC-UEA-I usually binds to the cytoplasm, nucleus and cell debris. In our experience, less than 20% of the samples can be optimally stained with FITC-UEA-I. It is necessary to explore a new method to improve the quality of fluorescence photomicrographs of late EPCs stained with FITC-UEA-I.
Here, we describe an updated protocol for the uptake of DiI-acLDL and the binding of FITC-UEA-I (Figure 1(right)). This method is simple and more efficient for obtaining good-quality fluorescence photomicrographs of FITC-UEA-I binding than previous methods.

| Isolation and culture of late EPCs
The study was approved by the hospital Ethics Committee. Human umbilical cord blood samples (20-30 ml) were collected from healthy newborns with informed consent. Late EPCs were cultured according to previously described techniques. 3,11,12 Briefly, total mononuclear cells (MNCs) were isolated from umbilical cord blood by Ficoll density gradient centrifugation. Cells were plated on six-well plates and maintained in endothelial cell growth medium-2 (EGM-2; Lonza, Walkersville, MD, USA). After 24 h of culture, the medium was changed for the first time, then changed daily for 7 days and finally changed every other day until the first passage. Late EPCs at passages 3-5 were used for fluorescent staining.

| Staining late EPCs with FITC-UEA-I after paraformaldehyde fixation
Late EPCs were plated on 48-well plates. After the cells had grown to 50-60% confluence, the cells were washed with PBS, fixed with 2% paraformaldehyde for 10 min, washed again for 5 min with PBS and then incubated with 20 μg/ml FITC-UEA-I for 1 h. After being washed with PBS, the cells were observed.
The cells were washed with PBS and fixed with paraformaldehyde when they reached 50-60% confluence. Then, the cells were washed again with PBS and observed.

| Updated protocol for double-staining late EPCs
Cell suspensions containing late EPCs, 15 μg/ml DiI-acLDL and 20 μg/ml FITC-UEA-I were seeded on 48-well plates. After the cells had grown to 50-60% confluence, late EPCs were washed with PBS and fixed with 2% paraformaldehyde for 10 min. After being washed again with PBS, late EPCs were analysed with a fluorescence microscope.

| Evaluation of immunofluorescence images of EPCs stained with FITC-UEA-I
Sixty-four samples of double-stained late EPCs were included in the present study. A total of 34 and 30 samples were stained with the typical and updated protocols respectively. An optimally stained immunofluorescence image was defined as binding of FITC-UEA-I to the cell membrane, no staining of the cytoplasm, nucleus, or cell debris and low background staining. The results were evaluated by two independent reviewers in a blinded manner.

| Statistical analysis
The data are presented as the number of samples. Differences between group means were assessed by chi-square tests using SPSS 24.0. p values <0.05 were considered significant.

| RE SULTS
MNCs had a round morphology after being isolated from human umbilical cord blood and plated on six-well plates (Figure 2A). After  Figure 4A,B). When late EPCs were incubated with FITC-UEA-I before fixation, the binding of FITC-UEA-I to the cytoplasm and nucleus was prevented ( Figure 4C). Based on these results, we updated the protocol for double-staining late EPCs (Figure 1 (right)).
In the present study, of 34 samples, only 6 samples (17.6%) were optimally stained with FITC-UEA-I (Table 1) by the typical protocol.
After using the updated protocol, 28 of 30 samples (93.3%) were optimally stained (Table 1) with FITC-UEA-I, with one sample failing to be stained and one sample that was not stained because of cell pollution. It is very easy to obtain good-quality fluorescence photomicrographs of late EPCs with this updated protocol ( Figure 5). Lectins are carbohydrate-binding proteins that can bind to specific carbohydrate residues, most of which are derived from plant species, invertebrates and higher animals. 18 UEA-I, a lectin isolated from Ulex europaeus, shows specific affinity for certain 1-fucose moieties and is a specific and sensitive marker for ECs. 17 It has been reported that UEA-I can bind to the cell membrane, cytoplasm and nucleus. 19,20 Fixation is an important step that is required for optimal immunofluorescence imaging. Paraformaldehyde fixation is commonly The results showed that incubation after paraformaldehyde fixation led to the binding of FITC-UEA-I to the cytoplasm and nucleus ( Figure 4A,B), which was prevented by incubation before paraformaldehyde fixation ( Figure 4C).
Based on these results, we adjusted the protocol (Figure 1  Obtaining good-quality fluorescence photomicrographs is easier with the updated protocol than with the typical method. Our data provide a promising double-staining protocol with DiI-acLDL and FITC-UEA-I for the primary characterization of late EPCs.

ACK N OWLED G EM ENTS
This work was supported by the Medical Scientific Research

CO N FLI C T O F I NTE R E S T
There are no conflicts of interest.