Flow Cytometry of the Side Population: Tips & Tricks

Abstract

Background: The Side Population (SP) has become an important hallmark for the definition of the stem cell compartment, especially in the detection of these cells and in their physical isolation by fluorescence-activated cell sorting (FACS). SP cells are CD34neg and were discovered using ultraviolet excitation based on the efflux of Hoechst 33342 (Ho342). Although the method works as originally described, we believe that this method is difficult for most investigators. First, because the ability to discriminate SP cells is based on the differential retention of Ho342 during a functional assay; second, because of the difficulties in setting the right experimental and acquisition conditions; and third, because the analysis of the acquired data requires an extensive expertise on flow cytometry to accurately detect the SP events. Methods: First of all and mainly for the SP application, the laser beam paths were exhaustively checked to ensure the lowest coefficients of variation. Blood suspensions were prepared by erythrocyte lysis with ammonium chloride and hematopoietic cells were labeled with Ho342. Results: The Ho342 concentration and the staining procedure are critical for the optimal resolution of the SP cells. Although UV laser alignment is very important to resolve the dim tail that outlines the SP, the problem with Ho342 excitation is not the Hoechst Blue emission, but rather the Hoechst Red's (because of the weak emission). Conclusions: Each laboratory must establish its own expected ranges based on its instrument and results may vary slightly due to instrument differences such as the narrowness of the band pass filters, laser power, laser emission wavelength, nozzle type, differential of pressure, light collection system (cuvette versus jet-in-air) and beam shaping optics.

1α,25‑Dihydroxyvitamin�D3 restrains stem cell‑like properties of ovarian cancer cells by enhancing vitamin�D receptor and suppressing CD44

“…Malignant MOSE (M-L) cells were separated by FACS into two populations. SP cells actively pump the Hoechst 33342 dye out of the cells, while NSP cannot and therefore this is a way to isolate these two cell populations (22)(23)(24)(25)(26). The percentage of SP cells in M-L cells reached up to 24.4% (Fig.…”

“…Our previous study demonstrated that the acquisition of stemness was closely associated with malignant transformation of MOSE cells (unpublished data). SP cells are an important hallmark for the definition of the stem cell-like characteristics (22)(23)(24)(25)(26); therefore, whether SP cells may be used to enrich ovarian CSCs was investigated. Malignant MOSE (M-L) cells were separated by FACS into two populations.…”

“…However, the effects of vitamin D 3 on ovarian CSCs remain largely unknown. Side population (SP) cells have been presented as functional markers of CSCs (22)(23)(24)(25)(26). The present study investigated whether 1α,25(OH) 2 D 3 was able to inhibit the stem cell-like phenotype of SP cells isolated from mouse OSE (MOSE) cells, and determined its possible underlying mechanisms.…”

NLR’s Analogs with Young Blood Cells in Monitoring of Toxicity of Long-Term Preventing Immunosuppression in the Liver Transplant’s Recipients

“…N. Shoutko, O. A. Gerasimova Open Journal of Biophysics cording to standard procedures to detect forms in the synthetic (S) and mitotic phases (M) of cell cycle [11] with Hoechst 33342 reagent (bisbenzimidazole fluorochrome; Sigma-Aldrich, St. Louis, Missouri, USA). CD133+, CD31+ cells, CD133+ AFP+, and CD31+ AFP+ double positive cells were stained using standard Miltenyi Biotec protocol for CD133/2 antibodies (Ab) conjugated with allophycocyanin (APC), BD Bioscience Pharmingen protocol for CD31 Ab with conjugated with fluorescein isothiocyanate (FITC), and R&D Systems protocol for α-fetoprotein (AFP) Ab conjugated with phycoerythrin (PE).…”

Redox-Regulation in Cancer Stem Cells

“…CSCs evade chemotherapeutic attacks by expressing highly efficient efflux pumps [ 3 ]. The rapid extrusion of Hoechst 33342 is an established FACS-based technique with which this CSC (which can be defined and represented in FACS as a so-called “side population”, SP) can be quantified and enriched by “sorting” [ 4 , 5 ]. The CSC phenotype is characterized by, among other things, the expression of various surface markers.…”