Assessment of Mitochondrial DNA Content and Mass in Macrophages

Materials and Reagents

1. Microcentrifuge tube (1.5 ml or 2 ml)
   
2. Lipopolysaccharide (InvivoGen, catalog number: tlrl-3pelps )
   
3. G-DEX^TM IIc Genomic DNA Extraction Kit (iNtRON Biotechnology, catalog number: 17231 )
   
4. Absolute or 70% ethanol (Merck Millipore Corporation, catalog number: 100983 )
   
5. Isopropanol (Sigma-Aldrich, catalog number: I9030 )
   
6. DEPC water (Merck Millipore Corporation, Calbiochem, catalog number: 9062-500 ML_CN )
   
7. Primer (EC21, Solgent Co)
   Stock concentration: 100 μM; Working concentration: 10 μM
   1. mND-1 (mitochondrially encoded NADH dehydrogenase 1)
      forward: 5’ GGCTACATACAATTACGCAAAG-3’
      reverse: 5’-TAGAATGGAGTAGACCGAAAGG-3’
      
   2. mPyruvate  kinase
      forward: 5’ ACTGGCCGGTGTCATAGTGA-3’
      reverse: 5’-TGTTGACCAGCCGTATGGATA-3’
      
8. SYBR green PCR master mix (QIAGEN, catalog number: 330520 )
   
9. MitoTracker Green FM (Thermo Fisher Scientific, Molecular Probes^TM, catalog number: M-7514 )
   
10. Accutase (Sigma-Aldrich, catalog number: A6964 )
    
11. Phosphate Buffered Saline (PBS) (Thermo Fisher Scientific, Gibco^TM, catalog number: 70011044 )
    
12. Formaldehyde for cell fixation (Sigma-Aldrich, catalog number: F8755 )
    
13. Triton^® X-100 (Sigma-Aldrich, catalog number: T8787 )
    
14. DMSO (Sigma-Aldrich, catalog number: D8418 )
    
15. DMEM (Thermo Fisher Scientific, catalog number: 11965 )
    
16. Macrophage Colony-Stimulating Factor (M-CSF) from mouse (Sigma-Aldrich, catalog number: M9170 )
    
17. Tris-HCl (Sigma-Aldrich, catalog number: T5941 )
    
18. EDTA solution (Sigma-Aldrich, catalog number: 03690 )
    
19. Heat-inactivated FBS (Thermo Fisher Scientific, Gibco^TM, catalog number: 1600044 )
    
20. Penicillin (50 U/ml)/Streptomycin (50 mg/ml) (Thermo Fisher Scientific, Gibco^TM, catalog number: 15140-122 )
    
21. TE buffer (see Recipes)
    
22. M-CSF-containing medium (see Recipes)
    

Equipment

1. Tabletop microcentrifuge (KITA, Hanil, model: Micro12 )
   
2. DNA electrophoresis chamber (Takara Bio Company, catalog number: AD140 )
   
3. Qiagen Rotor gene 6000 instrument for quantitative real-time PCR analysis (Takara Bio Company, catalog number: AD140)
   
4. BD FACSCanto^TM II flow cytometer (BD Biosciences)
   
5. 37 °C, 5% CO₂ incubator (Thermo Fisher Scientific, catalog number: 3111 )
   
6. Centrifuge (LaboGene, catalog number: i730R )
   
7. Water bath or heat block for heating at 65 °C
   
8. Vortex mixer (PRONEER, model: MX-S )
   

Software

1. FlowJo (Tree Star Inc)

Procedure