Be17 512f

The transcriptome analysis of the TCA/glycolytic genes of in vivo microglia was performed using our recently published data (Zhang et al., 2022)^{21 (link)}. In the study of Zhang et al., 2022 mice were given an intraperitoneal (ip) injection of 1 mg/kg LPS (Sigma‐Aldrich, Escherichia coli 011:B4,L4391) dissolved in DPBS (Lonza, BE17512F). Control mice received a respective volume of DPBS. After 3 h, animals were sacrificed under anesthesia and the brain was collected. Mouse microglia isolation from mouse were isolated from adult mouse brain using the protocol as described before⁶⁶ . Briefly, microglia-enriched percoll fractions were sorted by gating the DAPI^negCD11b^highCD45^intLy6c^neg cells. 200.000-300.000 microglia per brain were sorted for RNAseq experiments.

Male C57BL/6 mice (22–25 g; Envigo, the Netherlands) between 8 and 10 weeks of age were used for all experiments. Mice were raised on a 12‐hr light/dark cycle with food and water available ad libitum and were individually housed. All experiments were performed in the Central Animal Facility (CDP) of the UMCG, with protocol (15360‐03‐003) approved by the Animal Care and Use Committee (DEC) of the University of Groningen. Mice were given an intraperitoneal (ip) injection of 1 mg/kg lipopolysaccharide (LPS) (Sigma‐Aldrich, Escherichia coli 011:B4,L4391, Saint Louis, MD, USA) dissolved in DPBS (Lonza, BE17512F, Walkersville, MD, USA). Control mice received a respective volume of DPBS. After 3 hr, animals were sacrificed under anesthesia and the brain was collected.

Male C57BL/6J mice were obtained at the age of 7–9 weeks with weights in the range of 25–30 g (Envigo, Horst, The Netherlands). Upon arrival, a minimum acclimatization time of 2 weeks was ensured, where mice were monitored weekly in terms of general appearance and weight. All animals were housed individually to prevent fighting induced wounds and inflammation and randomly assigned to experimental conditions.
To induce endotoxin tolerance, mice received 1 mg/kg body weight LPS (Sigma-Aldrich, E. coli 0111:B4, L4391) diluted in dPBS (Lonza, BE17512F) to a total volume of 200 µL by intraperitoneal injection. Immediately following LPS administration, mice were housed in a recovery cabinet at 26 °C for 24 h. The weight and general health of injected animals were monitored daily until the body weight was completely restored (usually within 7 days), and monitoring was continued after recovery at a weekly basis. All control mice received 200 µL dPBS by intraperitoneal injection. After 4 weeks, the mice received a second injection with either dPBS or LPS (1 mg/kg body weight, diluted in 200 µL dPBS).

Microglia were isolated from whole brain (LPS) or spinal cord (EAE) as previously described in detail [21 (link)]. The whole isolation procedure was performed on ice. Mice were perfused with PBS (Lonza, BE17-512F) and CNS tissue was mechanically dissociated in HBSS (Gibco, 14,170–088) containing 0.6% glucose (Sigma-Aldrich, G8769) and 15 mM HEPES (Lonza, BE17-737E). Myelin was removed by 24.4% percoll (GE Healthcare, 17-0891-01) density gradient centrifugation at 950 g for 20 min at 4 °C. Fc receptors were blocked with anti-CD16/32 (5 μg/ml, clone 93, eBioscience, 14-0161-85), and cells were stained with anti-CD11b-APC-Cy7 (1 μg/ml, clone M1/70, eBioscience, A15390), anti-CD45-PE-Cy7 (1 μg/ml, clone 30-F11, eBioscience, 25-0451-82), anti-Ly6C-APC (1.5 μg/ml, clone HK1.4, Biolegend, 128,016), and anti-VISTA-PE (20 μg/ml, clone MIH63, Biolegend, 150,204) antibodies 30 min at 4 °C in HBSS without phenol red (Gibco, 14,175-053) containing 0.6% glucose, 15 mM HEPES, and 1 mM EDTA (Invitrogen, 15,575-020). Microglia were sorted on a MoFlo Astrios (Beckman Coulter) in siliconized tubes containing RNAlater (Qiagen, 76,104), centrifuged at 5000 g, and lysed in RLT + lysis buffer (Qiagen, 74,034).