Macs manual separator

To assess lung neutrophil IL5 mRNA levels in response to exogenous IL5, neutrophils were isolated from lung single-cell suspensions with the anti-Ly6G microbead kit and MACS manual separator (Miltenyi Biotec). The neutrophil mRNA was extracted using RNeasy Mini Kit and RNA reverse transcribed with iScript RT supermix. PCR amplification to detect IL5 mRNA expression was performed in an iTaq Universal SYBR Green Supermix using a Bio-Rad real-time system.
To assess with flow, neutrophils were isolated from mouse bone marrow as described [36 (link)]. Briefly, bone marrow cells were harvested from mouse femurs and tibias; 3 ml Histopaque 1119 was added in a 15 ml conical centrifuge tube and overlaid with 3 ml of Histopaque 1077, and this overlaid the bone marrow cell suspension. This was centrifuged at 872 × g at room temperature for 30 minutes. Neutrophils were collected from the interface of the Histopaque 1077 and Histopaque 1199 layers, then washed with RPMI 1640 (supplemented with 10% FBS) and centrifuged at 427 × g for 5 minutes at 4°C. 2 ng/ml or 10 ng/ml recombinant mouse IL5 was added into the separated neutrophils for 15 minutes, then PMA (10 ng/ml), ionomycin (500 ng/ml), and protein transport inhibitor (1 μl/ml) were added at 37°C for 3 hours, and neutrophil IL5 was detected by intracellular staining and flow cytometry.

LGR5⁺ AM epithelial cells were sorted by using magnetic Anti-LGR5 MicroBeads (Miltenyi Biotec) according to the manufacturer’s protocol. Briefly, cultured AM epithelial cells were labeled with Anti-LGR5 MicroBeads at 4 °C for 15 min. After washing, the cell suspension was applied to a LS Colum and separated with a magnetic MACS Manual Separator (Miltenyi Biotec). The purity of sorted LGR5⁻ and LGR5⁺ AM epithelial cells was examined by flow cytometry and confirmed by Western blot with a LGR5 antibody (Invitrogen, PA5-35304).