Ex vivo culture of SPMs
michela.deleidi, Federico Bertoli, Bianca Marchetti, Carmela Giachino
Abstract
Ex vivo culture of spleen derived macrophages.
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Steps
Ex vivo culture of spleen derived macrophages
Dissect spleens from abdominal cavity and filter it through a 40-μm nylon strainer.
Use red cell lysis buffer to remove red cells.
Then, obtain a single splenic cell suspension. Culture cells in Roswell Park Memorial Institute (RPMI) medium 1640 RPMI 1640 (BioConcept 1-41F01-I) supplemented with 10% FBS, 2millimolar (mM) L-Glutamine and antimicrobials (Penicillin-Streptomycin Pen 10'000 IU/ml Strep 10mg/mL and amphotericin B (250µg/mL BioConcept).
Culture mouse microglia BV2 cells from Elabscience (No.: EP-CL-0493) in parallel for each spleen culture preparation as controls.
Briefly, maintain BV2 cells in Roswell Park Memorial Institute (RPMI) medium 1640 supplemented (BioConcept 1-41F01-I) with 10% FBS (FBS-02-0500), 2millimolar (mM) L-Glutamine 5-10K50-H) and antimicrobials (Penicillin-Streptomycin Pen 10'000 IU/ml Strep 10mg/mL and amphotericin B (250µg/mL) (BioConcept 4-01F00-H).
Spleen macrophages (SPMs) differentiate into the M1 phenotype after stimulation with LPS (100 ng/ml) ± IFN-γ (10ng/mL).
For BV2 stimulation, replace RPMI by Dulbecco’s Modified Eagle Medium (DMEM) High Glucose. (BioConcept, 1-26F03-I).
