Cytometric bead array mouse flex kit

The blood drawn from eyeballs removed from anesthetized mice was centrifuged for 15 minutes at 300 × g. Then, the supernatant was stored at -80°C for subsequent analysis using a FACSCanto II flow cytometer (BD Biosciences, San Jose, CA, USA). Cytokines, such as interleukin (IL)-1β, IL-6, IL-10, IL-12, interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α), were quantified using a Cytometric Bead Array Mouse Flex Kit (BD Biosciences, San Jose, CA, USA). Annexin V-PE/ 7-AAD Apoptosis Detection Kit (BD Biosciences, San Jose, CA, USA) was used to identify and quantify apoptotic cells on a single-cell basis by flow cytometry, which contained Annexin V labeled with PE and 7-AAD. Simultaneous staining of cells with Annexin V-PE and the non-vital dye 7-aminoactinomycin D (7-AAD) allowed the discrimination of intact cells (Annexin V-PE negative, 7-AAD negative), early apoptotic (Annexin V- PE positive, 7-AAD negative) and late apoptotic or necrotic cells (Annexin V- PE positive, 7-AAD positive). 10×10⁴ cells were collected by flow cytometry in this experiment. The data obtained was analyzed using the FlowJo software (FlowJo, LLC, USA).

Levels of inflammatory cytokines were measured as previously described (Hod et al., 2010 (link)). Briefly, serum cytokines, including interleukin-6 (IL-6), tumor necrosis factor-α (TNF- α), monocyte chemoattractant protein-1 (MCP-1), and keratinocyte-derived chemokine (KC), were quantified using a Cytometric Bead Array Mouse Flex Kit (BD Biosciences). Data were analyzed using FlowJo software (Tree Star).