Purified Rat Anti-Mouse Siglec-F

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Bone marrow (BM), spleens, blood, and peritoneal cavity lavage cells were collected from 8–12 week old mice. Red blood cells were removed by lysis with an ammonium chloride-based buffer. Cells were stained with cocktails of antibodies	Cells were resuspended in PBS/2% FCS, 2 mM EDTA, 1 μg/ml propidium iodide (Sigma) to enable identification and exclusion of dead cells. Cells were analyzed on a BD LSR Fortessa X-20 flow cytometer (BD Biosciences).	Flow cytometric analyses were performed with FlowJo V10 software (FlowJo). Statistical tests and graphs were generated with Prism (GraphPad Software).

Upstream tips
Bone marrow (BM), spleens, blood, and peritoneal cavity lavage cells were collected from 8–12 week old mice. Red blood cells were removed by lysis with an ammonium chloride-based buffer. Cells were stained with cocktails of antibodies
Protocol tips
Cells were resuspended in PBS/2% FCS, 2 mM EDTA, 1 μg/ml propidium iodide (Sigma) to enable identification and exclusion of dead cells. Cells were analyzed on a BD LSR Fortessa X-20 flow cytometer (BD Biosciences).
Downstream tips
Flow cytometric analyses were performed with FlowJo V10 software (FlowJo). Statistical tests and graphs were generated with Prism (GraphPad Software).

Publication protocol

"Bone marrow (BM), spleens, blood, and peritoneal cavity lavage cells were collected from 8–12 week old mice. Red blood cells were removed by lysis with an ammonium chloride-based buffer. Cells were stained with cocktails of antibodies recognizing CD11b, IL5Rα, Siglec-F, B220, CD3, Ly6C, Ly6G, ST2, Lin, cKit, Scal, FcγRII/III, CD34, and CD45. Cells were resuspended in PBS/2% FCS, 2 mM EDTA, 1 μg/ml propidium iodide (Sigma) to enable identification and exclusion of dead cells. Cells were analyzed on a BD LSR Fortessa X-20 flow cytometer (BD Biosciences).

Cell populations were defined using the following surface markers: Eosinophils (CD11b+ Siglec-F+ IL5RαIntSSCHi), CMP (Lin-cKit + Sca1-CD34 + FcγRII/IIILo), GMP (Lin-cKit + Sca1-CD34 + FcγRII/III+), and EoP (Lin-cKit + Sca1-CD34 + FcγRII/III + IL5Rα+).

Flow cytometric analyses were performed with FlowJo V10 software (FlowJo). Statistical tests and graphs were generated with Prism (GraphPad Software)."

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