Anti mouse cd45 magnetic microbeads

Submandibular salivary glands were collected from B6 or B6DC mice. Single cell suspensions were isolated by mechanical or enzymatic disruption as previously described [32 ]. In short, salivary gland tissues were finely chopped and enzymatically digested with Collagenase type II (Worthington Biochemical Corporation, Lakewood Township, NJ, USA). Single cell suspensions were washed twice and subsequently filtered using nylon mesh and erythrocytes removed with red blood cell lysis buffer (Miltenyi Biotech, San Diego, CA, USA) prior to ex vivo magnetic cell isolation and flow cytometry. For the analyses of salivary gland epithelial cells, salivary gland epithelial populations were negatively selected by removing CD45+ leukocytes using anti-mouse CD45 magnetic MicroBeads (MiltenyiBiotec In short, salivary gland cell suspension was incubated with anti-mouse CD45 magnetic MicroBeads. These labeled cells were then passed through a magnetic separation column and washed three times, collecting the flow through for further flow cytometry and quantitative reverse transcription and PCR analyses.