Purified anti-mouse Ly-6G Antibody

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Single cell suspension of spleens and bone marrow (femurs and tibia) were prepared and filtered through a 70 μm cell strainer (BD Falcon). The primary cells were resuspended (107 cells/ml) in PBS containing 0.5%. BSA 0.5%.	Fc receptors were blocked with anti-CD16/CD32 for 15 min (5 μg/ml in PBS/BSA, clone 2.4G2, in house production). Subsequently, cells were washed with ice-cold PBS/0.5%BSA, and incubated with fluorescent labeled antibodies for 10 min on ice.	After washing twice, cells were re-suspended in PBS/0.5% BSA/2 mM EDTA, and analyzed in an LSRII flow cytometer (BD Biosciences). The resulting data were analyzed using the FlowJo software.

Upstream tips
Single cell suspension of spleens and bone marrow (femurs and tibia) were prepared and filtered through a 70 μm cell strainer (BD Falcon). The primary cells were resuspended (107 cells/ml) in PBS containing 0.5%. BSA 0.5%.
Protocol tips
Fc receptors were blocked with anti-CD16/CD32 for 15 min (5 μg/ml in PBS/BSA, clone 2.4G2, in house production). Subsequently, cells were washed with ice-cold PBS/0.5%BSA, and incubated with fluorescent labeled antibodies for 10 min on ice.
Downstream tips
After washing twice, cells were re-suspended in PBS/0.5% BSA/2 mM EDTA, and analyzed in an LSRII flow cytometer (BD Biosciences). The resulting data were analyzed using the FlowJo software.

Publication protocol

"Single cell suspension of spleens and bone marrow (femurs and tibia) were prepared and filtered through a 70 μm cell strainer (BD Falcon). The primary cells were resuspended (107 cells/ml) in PBS containing 0.5%. BSA 0.5%. In vitro generated CD115+ cells were generated as described below. To harvest them, culture supernatants were removed and cells were incubated with 2 mM EDTA for 10 min, and then harvested with cell scrapers.

Fc receptors were blocked with anti-CD16/CD32 for 15 min (5 μg/ml in PBS/BSA, clone 2.4G2, in house production). Subsequently, cells were washed with ice-cold PBS/0.5%BSA, and incubated with fluorescent labeled antibodies for 10 min on ice. After washing twice, cells were re-suspended in PBS/0.5% BSA/2 mM EDTA, and analyzed in an LSRII flow cytometer (BD Biosciences). The resulting data were analyzed using the FlowJo software."

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