Beckman navios flow cytometer

Peripheral blood, splenic and thymic cells (1 × 10⁶ cells in each case) from the MRL/lpr and Balb/c mice were stained to determine the number of CD4⁺CD25⁺Foxp⁺ Treg cells [fluorescein peridinin chlorophyll protein/PerCP-conjugated CD4⁺, allophycocyanin/APC-conjugated CD25⁺ (BioLegend, San Diego, CA, USA) and Alexa Fluor 488-conjugated Foxp⁺ (BD Pharmingen Corp., San Diego, CA, USA) T lymphocyte sub-populations], CD4⁺CD25^– Teff cells and IL-10⁺ Breg cells [(CD19⁺CD5⁺CD1d⁺ regulatory B cell) cocktail and Phycoerythrin/Cyanine Dye 7 (PE/Cy7)-conjugated anti-IL-10 antibody (BioLegend)] by flow cytometry (Beckman Navios flow cytometer, Beckman Coulter Inc., Brea, CA, USA).

Laboratory tests included white blood cell (WBC) count, neutrophils, lymphocytes, and serum cytokines, including interleukin (IL)‐1β, IL‐2, IL‐4, IL‐5, IL‐6, IL‐8, IL‐10, IL‐12p70, IL‐17, interferon (IFN)‐γ, IFN‐α, tumor necrosis factor (TNF)‐α. The routine blood parameters of the patients were measured by Sysmex XN 2000 automatic hematology analyzer, the blood biochemical parameters were measured by Cobas 8000 automatic biochemical immunoassay analyzer and its matching reagents, the serum inflammatory factors were measured by Beckman Navios flow cytometer, and the reagent kit was 12 cytokine detection kit (multiplex microsphere flow immunofluorescence method; Qingdao Ruisikai Biotechnology Co., Ltd.); the patients' elbow venous blood was collected and sent for testing immediately within 60 min after collection, and the testing was performed by laboratory doctors.

Desialylation of platelets was confirmed by lectin binding, using flow cytometric analysis. Fluorescein isothiocyanate (FITC)-conjugated Ricinus Communis agglutinin I (RCA-I; Vector Laboratories, Burlingame, CA, USA) was used for assessing platelet surface β-GlcNac exposure [8 (link), 13 (link)]. RCA-I at 5.0 µg/mL was added to each of isolated platelets after resuspension in buffer B. In the positive control group, 0.3 U/mL of sialidase was also added. The groups of platelets stored at 4 °C were then incubated at 37 °C for 20 min, using a WiseTherm^® HB-R (Daihan Sci., Seoul, Korea) water bath, and the groups of platelets stored at 22 °C were incubated with continuous gentle agitation. The lectin binding of platelets was analyzed using a Beckman Navios flow cytometer (Beckman Coulter Inc., Brea, CA, USA). Platelets were gated according to their forward scatter (FSC) and side scatter (SSC) characteristics.