Facslyric flow cytometry

Sample processing and flow cytometric analysis of mouse blood cells were performed as described elsewhere [23 (link)]. Briefly, an ethylenediaminetetraacetic acid–coated tube was used to collect mouse peripheral blood. Red blood cell lysis was performed by adding eBioscience 1X RBC Lysis Buffer (Invitrogen). The cells were blocked with Mouse BD FcBlock (BD Biosciences) and labeled with antibody cocktail (Supplementary Table 2). Sample acquisition was performed using BD FACSLyric flow cytometry (BD Biosciences), and data were analyzed using FlowJo software (version 10.8; Tree Star).

To determine the effects of vaccination on lymphocyte’s absolute number, the cell counts of T lymphocytes, B lymphocytes, NK cells and the subgroups (CD4+ or CD 8+) of T cells were determined by BD Multitest 6-color TBNK reagents on a BD FACSLyric flow cytometry (BD Bioscience, USA) according to the package insert. In brief, well-mixed whole blood samples were incubated with the 6-color TBNK reagents for 15 minutes in dark at 20-25°C in BD Trucount™ tubes before the red cells in the samples were lysed by adding the BD lysing solution. Then the samples were loaded and cell counts were measured on the flow cytometry and analyzed by BD FACSuite software.

Whole blood was collected into EDTA coated tube. For 1 ml whole blood, 9 ml of eBioscience™ 1X RBC Lysis Buffer (Invitrogen, Waltham, MA, USA) was added for red blood cells (RBCs) lysis, after 10 min, cells were centrifuged and resuspended in flowcytometry buffer (3% Heat inactivated FCS, 1 mM EDTA, 10 mM HEPES, 0.09% NA acid), 2 million cells were blocked with 2 µl Mouse BD FcBlock™ (BD Biosciences) for 5 min on ice, cells were resuspended in antibody cocktail for 20 min after centrifugation, then, washed twice with cold PBS and stained with FITC Annexin V kit (Biolegend, San Diego, CA, USA) and 7-AAD (Invitrogen, Waltham, MA, USA) according to manufacturer’s instructions. Antibodies used in the antibody cocktail can be found in Supplementary Table 1. Samples were acquired by using BD FACSLyric™ flow cytometry (BD Biosciences). UltraComp eBeads™ Compensation Beads (Invitrogen, Waltham, MA, USA) were used to set up the compensation. Fluorescence minus one (FMO) samples were used to identify negative population for each antibody. Data analyze were performed by using FlowJo software (version 10.8; Tree Star, Ashland, USA).