PMID- 35723458
OWN - NLM
STAT- MEDLINE
DCOM- 20220622
LR  - 20240320
IS  - 1940-087X (Electronic)
IS  - 1940-087X (Linking)
IP  - 183
DP  - 2022 May 31
TI  - Isolation and In vitro Culture of Bone Marrow-Derived Macrophages for the Study 
      of NO-Redox Biology.
LID - 10.3791/62834 [doi]
AB  - Macrophages are derived from hematopoietic progenitor cells throughout the body, 
      are central to inflammatory processes, and participate in innate and adaptive 
      immune responses. In vitro study of macrophages can be undertaken by ex vivo 
      culture from the peritoneum or through differentiation of myeloid bone marrow 
      progenitor cells to form bone marrow-derived macrophages (BMDMs). A common 
      approach to macrophage differentiation from precursors involves the use of 
      conditioned media from L929 cells (LCM). This media is easy to self-produce but 
      suffers from batch variability, and its constituents are undefined. Similarly, 
      Foetal Bovine Serum (FBS) is used to support growth but contains a vast mixture 
      of undefined molecules that may vary between batches. These methods are not 
      adequate for the study of nitric oxide biology and redox mechanisms as they both 
      contain substantial amounts of small molecules that either interfere with redox 
      mechanisms or supplement levels of cofactors, such as tetrahydrobiopterin (BH4), 
      required for the production of NO from inducible nitric oxide synthase (iNOS). In 
      this report, we present an optimized protocol allowing for control of the 
      NO-redox environment by reducing the levels of exogenous biopterin while 
      maintaining conditions suitable for cell growth and differentiation. Tight 
      control of culture media composition helps ensure experimental reproducibility 
      and facilitates accurate interpretation of results. In this protocol, BMDMs were 
      obtained from a GTP cyclohydrolase (GCH)- deficient mouse model. Culture of BMDMs 
      was performed with media containing either (i) conditioned LCM, or (ii) 
      recombinant M-CSF and GM-CSF to produce minimal artifacts while obtaining BH4 and 
      NO-deficient culture conditions - thus allowing for the reproducible study of 
      NO-redox biology and immunometabolism in vitro.
FAU - Diotallevi, Marina
AU  - Diotallevi M
AD  - BHF Centre of Research Excellence, Division of Cardiovascular Medicine, Radcliffe 
      Department of Medicine, John Radcliffe Hospital, University of Oxford; Wellcome 
      Centre for Human Genetics, University of Oxford.
FAU - Nicol, Thomas
AU  - Nicol T
AD  - BHF Centre of Research Excellence, Division of Cardiovascular Medicine, Radcliffe 
      Department of Medicine, John Radcliffe Hospital, University of Oxford; Wellcome 
      Centre for Human Genetics, University of Oxford.
FAU - Ayaz, Faseeha
AU  - Ayaz F
AD  - BHF Centre of Research Excellence, Division of Cardiovascular Medicine, Radcliffe 
      Department of Medicine, John Radcliffe Hospital, University of Oxford; Wellcome 
      Centre for Human Genetics, University of Oxford.
FAU - Bailey, Jade
AU  - Bailey J
AD  - BHF Centre of Research Excellence, Division of Cardiovascular Medicine, Radcliffe 
      Department of Medicine, John Radcliffe Hospital, University of Oxford; Wellcome 
      Centre for Human Genetics, University of Oxford.
FAU - Shaw, Andrew
AU  - Shaw A
AD  - BHF Centre of Research Excellence, Division of Cardiovascular Medicine, Radcliffe 
      Department of Medicine, John Radcliffe Hospital, University of Oxford; Wellcome 
      Centre for Human Genetics, University of Oxford.
FAU - McNeill, Eileen
AU  - McNeill E
AD  - BHF Centre of Research Excellence, Division of Cardiovascular Medicine, Radcliffe 
      Department of Medicine, John Radcliffe Hospital, University of Oxford; Wellcome 
      Centre for Human Genetics, University of Oxford.
FAU - Davies, Ben
AU  - Davies B
AD  - BHF Centre of Research Excellence, Division of Cardiovascular Medicine, Radcliffe 
      Department of Medicine, John Radcliffe Hospital, University of Oxford; Wellcome 
      Centre for Human Genetics, University of Oxford.
FAU - Channon, Keith M
AU  - Channon KM
AD  - BHF Centre of Research Excellence, Division of Cardiovascular Medicine, Radcliffe 
      Department of Medicine, John Radcliffe Hospital, University of Oxford; Wellcome 
      Centre for Human Genetics, University of Oxford.
FAU - Crabtree, Mark J
AU  - Crabtree MJ
AD  - BHF Centre of Research Excellence, Division of Cardiovascular Medicine, Radcliffe 
      Department of Medicine, John Radcliffe Hospital, University of Oxford; Wellcome 
      Centre for Human Genetics, University of Oxford; mark.crabtree@well.ox.ac.uk.
LA  - eng
GR  - RG/17/10/32859/BHF_/British Heart Foundation/United Kingdom
GR  - PG/15/35/31403/BHF_/British Heart Foundation/United Kingdom
GR  - CH/16/1/32013/BHF_/British Heart Foundation/United Kingdom
GR  - RG/12/5/29576/BHF_/British Heart Foundation/United Kingdom
GR  - 090532/Z/09/Z/WT_/Wellcome Trust/United Kingdom
GR  - FS/14/56/31049/BHF_/British Heart Foundation/United Kingdom
GR  - RE/18/3/34214/BHF_/British Heart Foundation/United Kingdom
GR  - RE/13/1/30181/BHF_/British Heart Foundation/United Kingdom
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Video-Audio Media
DEP - 20220531
PL  - United States
TA  - J Vis Exp
JT  - Journal of visualized experiments : JoVE
JID - 101313252
RN  - 31C4KY9ESH (Nitric Oxide)
SB  - IM
MH  - Animals
MH  - Biology
MH  - *Macrophages
MH  - Mice
MH  - *Nitric Oxide
MH  - Oxidation-Reduction
MH  - Reproducibility of Results
EDAT- 2022/06/21 06:00
MHDA- 2022/06/23 06:00
CRDT- 2022/06/20 09:03
PHST- 2022/06/20 09:03 [entrez]
PHST- 2022/06/21 06:00 [pubmed]
PHST- 2022/06/23 06:00 [medline]
AID - 10.3791/62834 [doi]
PST - epublish
SO  - J Vis Exp. 2022 May 31;(183). doi: 10.3791/62834.