Beckman cytometer

Cellular apoptosis was detected using an Annexin V-allophycocyanin (APC)/7-amino-actinomycin D (7-ADD) kit (KeyGENE, China). In this study, flow cytometry was used to analyze cellular apoptosis. The RNP@CaP-TAT and RNP@CaP-TAT-control cell samples were re-suspended with 500 µl 1 × binding buffer at a concentration of (1–3) × 10⁶ cells/ml. Subsequently, Flow cytometry was routinely performed according to the manufacture’s protocol. After incubation with reagents from the indicated assay kit, cells were analyzed using a Beckman cytometer (Beckman Coulter, United States) and CytExpert software (Beckman Coulter, United States). The cells were divided into four regions (Q1–Q4). Region Q1 was representative of mechanical error; region Q2 was representative of late apoptotic or necrotic cells; region Q3 was representative of early apoptotic cells; and region Q4 was representative of living cells.

Dexamethasone (DEX) was extensively applied as the first-line treatment of ITP patients, which may correct the imbalance of T cell subsets. For the apoptosis analysis, PBMCs cultured with or without 10 mmol/L DEX after NLRP3 activation or not were washed with PBS twice and stained with Alexa Fluor 488 Annexin V and PI using the Alexa Beyotime Cell Apoptosis Kit (Beyotime, China) according to the manufacturer's protocol. Early apoptosis (Annexin-positive and PI-negative cells) and late apoptosis (Annexin-positive and PI-positive cells) were counted, respectively, using a Beckman cytometer within 15 min after being stained. Data analysis was carried out using the Kaluza analysis software.

All data acquisition was performed using a Beckman cytometer. The CytExpert software was used for data analysis and graphical representation. Basically, BMDMs were stimulated with PBS and LPS (100 ng/ml) with or without lactic acid (15 mM) for 24 h. For fluorescence-activated cell sorting (FACS) analysis, BMDMs were collected and stained with the manufacturer’s suggested concentrations of FITC anti-CD11b, PE anti-F4/80, and APC anti-CD86 for 15 min at room temperature in the dark. For intracellular staining, the cells were stained with FITC anti-CD11b and PE anti-F4/80, fixed in a fixation buffer (BioLegend) for 20 min, and resuspendend with intracellular staining permeabilizaiton buffer. Finally, the cells were incubated with APC anti-CD206 and quantified.