Jun 13, 2023

Public workspaceEx vivo culture of SPMs

DOI
dx.doi.org/10.17504/protocols.io.8epv5jpdjl1b/v1
  • 1Mitochondria and Inflammation in Neurodegenerative Diseases, DZNE, Tübingen-Germany;
  • 2Hertie Institute for Clinical Brain Research, University of Tübingen;
  • 3Neuropharmacology Laboratory, Oasi Research Institute-IRCCS, Troina, Italy;
  • 4Biomedical and Biotechnological Sciences, Pharmacology Section, University of Catania-Italy
carmela giachino
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DOI: dx.doi.org/10.17504/protocols.io.8epv5jpdjl1b/v1
Protocol Citation: michela.deleidi, Bianca Marchetti, Federico Bertoli, Carmela Giachino 2023. Ex vivo culture of SPMs. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5jpdjl1b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 31, 2023
Last Modified: June 13, 2023
Protocol Integer ID: 79805
Keywords: Ex vivo culture, spleen derived macrophages
Funders Acknowledgement:
ASAP-Aligning Science Across Parkinson’s
Grant ID: ASAP-000420
Abstract
Ex vivo culture of spleen derived macrophages.
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Materials
Materials

ReagentRPMI 1640Coris bioConceptCatalog #1-41F01-I

ReagentDMEM High Glucose (45g/l)Coris bioConceptCatalog #1-26F03-I

  • 10% FBS
  • 2mM L-Glutamine
  • Penicillin-Streptomycin Pen 10'000 IU/ml
  • amphotericin B (BioConcept 4-01F00-H)
  • microglia BV2 cells from Elabscience (No.: EP-CL-0493)
Ex vivo culture of spleen derived macrophages
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, Concentration2 millimolar (mM) L-Glutamine and antimicrobials (Penicillin-Streptomycin Pen 10'000 IU/ml Strep Amount10 mg/mL and amphotericin B (Amount250 µg/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), Concentration2 millimolar (mM) L-Glutamine 5-10K50-H) and antimicrobials (Penicillin-Streptomycin Pen 10'000 IU/ml Strep Amount10 mg/mL and amphotericin B (Amount250 µg/mL ) (BioConcept 4-01F00-H).
Spleen macrophages (SPMs) differentiate into the M1 phenotype after stimulation with LPS (100 ng/ml) ± IFN-γ (Amount10 ng/mL ).
For BV2 stimulation, replace RPMI by Dulbecco’s Modified Eagle Medium (DMEM) High Glucose. (BioConcept, 1-26F03-I).