Moflo xdp cell sorter

HCC stem-like cells were collected for functional assays using SP cells or CD133-positive cells from the MHCC-97L cell line sorted by flow cytometry.
The SP analysis procedures were based on a previously described protocol [27 (link)]. Briefly, the cells were trypsinized and suspended in DMEM with 2% FBS and 10 mM HEPES buffer. Next, the cells were stained for 90 min with 5 μg/ml Hoechst 33342 dye (Invitrogen, USA) in the presence or absence of 50 μM verapamil. The incubation was performed with shaking at intervals. After the cells were incubated, they were resuspended in ice-cold PBS containing 2 μg/ml PI. Flow cytometric analysis was performed using a MoFlo XDP cell sorter (Beckman Coulter, Fullerton, CA). The SP gate was defined as the diminished area on the dot plot in the presence of verapamil. The SP MHCC-97L cells were used to evaluate the effects of pimozide.
The CD133-positive population in the MHCC-97L cell line was sorted by flow cytometry using a MoFlo XDP cell sorter (Beckman Coulter, Fullerton, CA). The cells were stained with PE-conjugated anti-human CD133/1 (clone AC133-MAC, Miltenyi Biotec, Auburn, CA, USA). Isotype-matched mouse immunoglobulins served as controls. The CD133-positive cells in the MHCC-97L cell line were used for further functional analysis.

Cells were seeded in triplicate wells of 6-well plates at 1 × 10⁵ cells per well, treated with cinobufagin, and collected through trypsinization. The cell pellets were washed twice in PBS before the following analyses. All experiments were performed three times and the results were presented as mean ± SD.
For cell cycle analysis, cell pellets were fixed in 70% ice-cold ethanol at − 20 °C for 1 h. After washing in PBS twice, cells were stained with working solutions from the Cell Cycle Detection kit (BestBio, Shanghai, China). Samples were loaded onto and analyzed by the MoFlo XDP Cell Sorter (Beckman Coulter, Indianapolis, IN, USA). Data were processed with the FlowJo software (FlowJo, Ashland, OR, USA).
For the analysis of apoptosis, cells were resuspended in a binding buffer from the Annexin V-FITC Apoptosis Detection kit (BestBio) according to the instructions provided by the manufacturer. The cells were loaded onto and analyzed by the MoFlo XDP Cell Sorter (Beckman Coulter) and data were processed with the CytExpert software (Beckman Coulter).

RBC-lysed single-cell suspensions were stained in ice-cold PBS containing LIVE/DEAD fixable near-IR dead cell stain (Life Technologies) and the appropriate pretitered Abs. Cell numbers in the bone marrow are quoted per leg (one femur and one tibia). B10 cells were purified using the Miltenyi Biotec Regulatory B cell isolation kit with 24-h in vitro stimulation followed by flow cytometric sorting for B220⁺CD19⁺IL-10⁺ cells. To isolate plasma cells and plasmablasts, we enriched organ suspensions from mice immunized 5 d earlier with SRBC for CD138⁺ cells by staining with anti–CD138-PE and then using anti-PE beads (Miltenyi Biotec). To isolate germinal center B cells, we depleted splenocytes from mice immunized 10 d earlier with SRBC using anti–CD43-biotin and anti–IgD-biotin followed by streptavidin-Dynabeads (Life Technologies) and then sorted for B220⁺PNA⁺GL7⁺Fas⁺CD38^low. Data were collected on a BD FACSCanto II or sorted on BD FACSAria II, BD Influx, or Beckman Coulter MoFlo XDP cell sorters. Data were analyzed using FlowJo 9.6 (TreeStar).

The pRetroX-IRES-ZsGreen1-TRAPPC6AΔ retroviral plasmid and an insertless pRetroX-IRES-ZsGreen1 control retroviral vector were independently transfected into the AmphoPack-293 packaging cell line (Clontech) by using Lipofectamine LTX and Plus reagents. Viral supernatants from transfectants were used to transduce A549 cells. After 48 h, transduction was repeated to enrich for transductants. GFP-positive cells were then harvested 48 h later by using a MoFlo XDP cell sorter (Beckman Coulter), propagated, and examined for TRAPPC6AΔ overexpression by Western blotting. To study the effect of TRAPPC6AΔ overexpression on the growth of influenza virus, the WSN virus was used to infect the TRAPPC6AΔ-overexpressing A549 cell line or an A549 control cell line transduced with empty retrovirus at an MOI of 0.01. Supernatants were collected at 24 and 48 h p.i., and virus titers were determined by plaque assays on MDCK cells.

Cells were seeded at a density of 2.5 × 10⁵ per well in the 6-well dishes. Cells were incubated with 2 μM C11-BODIPY-581/591 (C11-BODIPY) (D3861; Thermo Fisher Scientific, Waltham, MA, USA) for 15 min or with 5 μM 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) (S0033S; Beyotime Biotechnology, Shanghai, China) for 30 min after receiving different drug treatments. Cells were then digested with trypsin and resuspended in phosphate-buffered saline (PBS). For DCFH-DA and C11-BODIPY staining, the data were collected using the MoFlo XDP cell sorter (Beckman Coulter, Indianapolis, IN, USA) from FL1. A minimum of 10,000 cells per treatment group were analyzed with CytExpert (Beckman Coulter, Brea, USA) or FlowJo (FlowJo, LLC, Ashland, OR, USA) software.