Fresh breast milk was spiked with murine BMDC at a final concentration of 0.75×106 cells/ml or was left unspiked. Milk was divided into equal volumes and was either processed immediately or stored at −80°C, 4°C, RT or 37°C for the indicated time periods. Breast milk was centrifuged at 3,000 g for 10 minutes. Cell pellets were resuspended and washed twice in IMDM containing 5% FCS and centrifuged at 500 g for 10 minutes. Cell pellets were resuspended in PBS/1% FCS. Cellular viability was determined by trypan blue exclusion.
Alternatively, cells were incubated with APC-conjugated anti-mouse MHC class II (M5/114, eBioscience) or the corresponding isotype control, or Alexa488-conjugated Annexin V (Life Technologies, Bleiswijk, Netherlands) and propidium iodide (PI; eBioscience) as indicated by manufacturer. Cells were analyzed using a FACS Canto II and FACS Diva software (BD Biosciences), and FCS Express software (De Novo software, Los Angeles, CA, USA). Murine cell viability was determined by gating on MHC class II positive cells, and determining the percentage of Annexin V and/or PI positive cells.
Alternatively, cells were incubated with APC-conjugated anti-mouse MHC class II (M5/114, eBioscience) or the corresponding isotype control, or Alexa488-conjugated Annexin V (Life Technologies, Bleiswijk, Netherlands) and propidium iodide (PI; eBioscience) as indicated by manufacturer. Cells were analyzed using a FACS Canto II and FACS Diva software (BD Biosciences), and FCS Express software (De Novo software, Los Angeles, CA, USA). Murine cell viability was determined by gating on MHC class II positive cells, and determining the percentage of Annexin V and/or PI positive cells.