M-CrenegSirt1flox/flox) mice. All animals used in experiments
are aged from 6 to 12 weeks.
2.L. infantum(MHOM/MA/67/ITMAP-263) promastigotes.2.2 Culture Reagents 1. Macrophage medium: DMEM, 4.5 g/L glucose, 20 mM
HEPES, 10% (v/v) heat-inactivated fetal bovine serum (FBS),
2mML-glutamine, 100 U/ml penicillin, 100 mg/ml
streptomycin.
2.Leishmaniamedium: RPMI 1640, 20 mM HEPES, 10% (v/v)
heat-inactivated FBS, 2 mML-glutamine, 100 U/ml penicillin,
100 mg/ml streptomycin.
- Macrophage growth factor: 100μg/ml M-CSF.
- XF medium: unbuffered DMEM, 4.5 g/L glucose, 2% (v/v)
FBS, 2 mML-glutamine, 100 U/ml penicillin, 100 mg/ml
streptomycin. - Carboxyfluorescein succinimidyl ester (CFSE, FITC) and
eFluor670 (APC): 5 mM stock solution. - AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide).
- Compound C (also known as Dorsomorphin).
- SRT1720.
2.3 FACS Staining 1. DMEM-EDTA: DMEM, 50 mM EDTA.
- Phosphate buffer saline (PBS): 145 mM NaCl, 2.7 mM KCl,
1.5 mM KH 2 PO 4 , 8 mM Na 2 HPO 4 ·2H 2 O, pH 7. - PBS-FBS: PBS, 2% (v/v) FBS.
- Probes: 14.6 mM 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)
amino]-2-deoxy-D-glucose (2-NBDG) and 1 mg/ml
7-Aminoactinomycin D (7-AAD).
2.4 Antibodies 1. FACS: anti-mouse monoclonal antibodies—anti-CD11b-PE
(M1/70), anti-Ly6C-PerCP/Cy5.5 (HK1.4), anti-Ly6G-
Pacific Blue (1A8), and anti-F4/80-PerCP-Cy5.5 (BM8).
- Cell magnetic separation: CD3εand CD19 microbeads.
- PBS-EDTA buffer: PBS, 0.5% (w/v) BSA (bovine serum albu-
min), 2 mM EDTA, pH 7.2. - Immunoblot: total AMPKα(23A3), AMPKαphosphorylated
at Thr172, total SIRT1 (H-300), total PGC1β(E-9),β-actin
(C4), total PGC1α (4C1.3), and horseradish peroxidase-
coupled secondary reagents.
2.5 Quantitative PCR
Analysis
- TRIzol reagent.
- Chloroform.
- Isopropanol.
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