Coulter fc500 flow cytometer

The cellular uptake of the CDs‐PEI‐AS1411 and the CDs‐PEI was further studied with flow cytometry. MCF‐7 and L929 cells were cultured overnight at 2 × 10⁵ cells per well in 6‐well plates. After that, the cells were treated with CDs‐PEI‐AS1411 or CDs‐PEI (the CDs concentration of 200 µg/mL) for 6 or 12 hours. Then, the cells were washed for three times with PBS and centrifuged at 1000 rpm for 5 minutes. After resuspended in 500 µL PBS, the cells were detected by a Coulter FC500 flow cytometer (Beckman).

PBMCs were isolated from heparinized venous blood sample by Ficoll-Histopaque (Sigma-Aldrich) density-gradient centrifugation. Cells were then harvested and washed twice and stained for 20 min at 4°C using specific antibodies. To identify transitional, mature, and primarily memory B cell subpopulations, cells were stained with the combination of the following monoclonal antibodies: PC5-labelled anti-CD19 (Beckman Coulter), Alexa Fluor 488-labelled anti-CD24 (BioLegend), and PC7-labelled anti-CD38 (BD Biosciences). After 30 min incubation, leucocytes were washed in phosphate-buffered saline (PBS) supplemented with bovine serum albumin (BSA) (10 g/L) and sodium-azide (2 mg/L). The cells were fixed subsequently using 500 μL of 1% paraformaldehyde. Mouse immunoglobulin (Ig) G1 antibodies were used as isotype control throughout the experiments. Samples were processed according to the Coulter Q-PREP protocol and system (Beckman Coulter Inc., Miami, FL, USA). Measurements were performed on a Coulter FC500 flow cytometer (Beckman Coulter Inc.). CD19⁺CD24^highCD38^high (transitional), CD19⁺CD24^intCD38^int (mature), and CD19⁺CD24^highCD38⁻ (primarily memory) B cells were quantified as their percentage in the CD19⁺ lymphocyte population.

Heparinized blood samples collected from different groups were dual- or triple-stained with anti-human CD3 (clone UCHT1, Beckman Coulter, Fullerton, CA), anti-human CD4 (clone SFCI12T4D11, Beckman Coulter), anti-human CD8 (clone SFCI21Thy2D3, Beckman Coulter), anti-human CD16 (clone 3G8, Beckman Coulter), anti-human CD19 (clone 89B, Beckman Coulter) and anti-human CD56 (clone N901, Beckman Coulter) mAbs conjugated with phycoerythrin (PE), fluorescein isothiocyanate (FITC) or phycoerythrin-Texas Red (ECD). PE-, FITC- or ECD-conjugated anti-human isotype-matched mAbs (Beckman Coulter) were used as negative controls. Erythrocytes were lysed with OptiLyse C (Beckman Coulter). FACScan analysis was performed for at least 10,000 events for detection of lymphocyte subsets in the peripheral blood including CD3⁺, CD3⁺CD4⁺, CD3⁺CD8⁺, CD3⁻CD19⁺ and CD3⁻CD16⁺CD56⁺ cells on a Coulter FC500 flow cytometer (Beckman Coulter) equipped with EXPO32 software (Beckman Coulter).

Zeta potential analysis and dynamic light scattering (DLS) analysis were conducted on ZetaPlus 90 Plus/BI-MAS (Brook haven, USA). Transmission electron microscopic (TEM) images were captured on JEM-2800 transmission electron microscope (JEOL Ltd., Japan). MTT and ELISA assays were conducted on Victor X3 Multimode Plate Reader (PerkinElmer, USA). The gel electrophoresis was performed with Mini-Protean Tetra Cell and PowerPac™ Basic Power Supply (Bio-Rad, USA). The intracellular fluorescence images were acquired through TCS SP5 confocal laser scanning microscope (CLSM) (Leica, Germany). Flow cytometry was accomplished on Coulter FC-500 flow cytometer (Beckman-Coulter, USA). The UV–Vis absorption spectra were recorded with UV-3600N UV/VIS spectrophotometer (Shimadzu, Japan). The fluorescence spectra were recorded with F-4700 spectrofluorophotometer (Hitachi, Japan).

Ten to fifteen milliliters of heparinized peripheral blood were collected for CIK in vitro expansion from health volunteers with informed consent. Peripheral blood mononuclear cells (PBMC) were separated by Ficoll density gradient centrifugation (GE Healthcare) and seeded at a concentration of 1.5×10⁶ cells/ml in culture medium (Gibco, Life Technologies) with the addition of 1000 U/ml interferon-γ (Pepro Tech) and 50ng/ml anti-human OKT-3 (eBioscience) on day 0 and 300U/ml interleukin (IL)-2 (Huaxin High Biotechnology, Shanghai) on day 1. Phenotype and immune profile of CIK cells were characterized by multiparameter flow cytometric analysis using following monoclonal antibodies: CD3-FITC, CD56-PC7, CD3-PC7, CD4-FITC, CD8-FITC, CD314-PC7 (anti-NKG2D), CD152-PE (anti-CTLA-4), CD223-APC (anti-LAG-3), CD226-PE (anti-DNAM-1), CD244-FITC (anti-2B4), CD274-PE (anti-PD-L1), CD279-APC (anti-PD-1), Foxp3-PE (mAbs were from BD Biosciences). Flow cytometric analysis was performed using the Coulter FC500 flow cytometer (Beckman Coulter). Data were further analyzed by Flow Jo 7.6.5 software (Tree Star).

Neurons were processed for fluorescence activated cell sorting (FACS) analysis as described previously [8 (link)]. DNA content and ploidy were assessed by using a Coulter FC500 flow cytometer (Beckman Coulter, Brea, CA, USA), and cell cycle distribution profiles were analyzed with the ModFit software program. Apoptotic neurons were scored from the area of hypoploid DNA preceding the G0/G1 DNA peak. A typical cell cycle distribution profile of control neurons and neurons after exposure to Aβ_(1-42) oligomers or Aβ_(1-42) monomers as a negative control is shown in Supplementary Fig. S3.

Five- to six-week-old female NOD/SCID mice were specially reared without pathogens in the Experimental Animal Center of Chongqing Medical University. Mice received sub-lethal radiation of 250 cGy before injection. After 2–3 h, mice were injected through the tail vein with 5 × 10⁶ shRNA-transformed K562/G01 cells in 200 μL PBS. The control group (PBS) received sub-lethal radiation of 250 cGy and then injected with 200 μl of cell-free PBS. The body weight, white blood cell count (WBC) and mental state of mice were monitored weekly. For WBC count, 10 μl of peripheral blood was collected and dissolved in 190 μl of 3% glacial acetic acid using the tail cutting method. After standing on ice for 10 min, peripheral blood WBC count was determined [17 (link)]. BM and peripheral blood from xenograft mice were treated with erythrocyte lysis buffer (Biolegend, USA). The GFP⁺ % of transduced K562/G01 cells was calculated using Coulter FC500 flow cytometer (Beckman, CA, USA) and analyzed using the FlowJo software. All animal experiments were conducted following the Ethical Guidelines for Animal Experiment Institutions and approved by the Ethics Committee of Chongqing Medical University.