Beckman flow cytometry

24 h after transfection, the HeLa cells were digested with trypsin to prepare single cell suspension. Then, the cells were gently washed with precooled PBS 3 times, and the cell concentration was maintained at 3 × 10⁵ cell/ml. The cells were then treated with annexin V-FITC & PI apoptosis detection kit (Sangon, China). After treatment, the number of apoptotic cells analyzed by Beckman flow cytometry (Beckman, United States).

The study was approved by the Institutional Ethics Committee of The Third Outpatient Department, General Hospital of Northern Theater Command on February 1, 2017 (No.[2017]015), and written informed consent was obtained from all participants.
Two mililiter and 1mL of fasting peripheral venous blood were collected from the enrolled children twice in the morning by vacuum anticoagulant tube. Again two mililiter peripheral blood samples were taken for processing within four hour. The steps were as follows: post-stimulation of cells → labeling tube → fixation/rupture of membranes and cell perforation → intracellular immunofluorescence labeling, and then Beckman flow cytometry (Beckman-Coulter, Inc., United States) was utilized to detect the expression levels of T lymphocyte subsets (CD₃⁺, CD₄⁺, CD₈⁺, CD₄⁺/CD₈⁺) in peripheral blood. Meanwhile, regulatory T cells (Treg) and helper T lymphocytes (Th1, Th2, Th17) in CD4⁺ subsets were detected, and Th1/Th2 value was calculated.

The stained peripheral blood mononuclear cells were tested by Beckman flow cytometry (Beckman Coulter, Inc., California, USA). The specific steps were as follows: first, we mixed the biotinylated SARS-CoV-2 spike RBD protein (40592-V08H2-B, Sino biological, Beijing, China) with streptavidin-BV421 (405225, Biolegend, California, USA) in a 4:1 mole ratio and leave for 1 h to get the antigen probe; second, peripheral blood mononuclear cells were obtained from whole heparinized blood; the density gradient centrifugation was performed by Histopaque (10771, Sigma–Aldrich, St Louis, Missouri, USA) and then cleaned by cytometric staining buffer (FACS, 2%FBS) cells, besides we added antigen probe (1:33.3), fluorescence-coupled antibody [anti-human IgG Fc (410722, Biolegend), anti-human IgM (314524, Anti-human) CD3 (300430, Biolegend), anti-human CD19 (302212, Biolegend), anti-human CD21 (354918, Biolegend), and anti-human CD27 (356406, Biolegend)] into the cells and dyed at 4°C for 30 min under dark condition. After being re-suspended with FACS buffer, the samples were tested on the machine. The data were analyzed by Flow Jo software (V10.0.7).