2-nbdg glucose uptake cell-based assay kit  (Cayman Chemical)

Journal: Advanced Science

Article Title: Flipping the Switch: MeCP2‐Mediated Lactylation Rewires Microglial Metabolism and Inflammation via the HK2/mTOR Axis in Poststroke Neuroinflammation

doi: 10.1002/advs.202513400

Figure Lengend Snippet: MeCP2 K210 lactylation drives microglial metabolic reprogramming and inflammatory activation, suppressed by K210R mutation. A) Flow cytometry of intracellular calcium levels (Fluo‐4) in murine BV2 cells expressing vector, wild‐type (WT), or K210R‐mutant MeCP2. B) MitoTracker Deep Red staining and quantification of mitochondrial membrane depolarization. C) MitoSOX‐based measurement of mitochondrial ROS. D) Glucose uptake assessed via 2‐NBDG fluorescence. E,F) Enzymatic activity of pyruvate dehydrogenase (PDH, E) and pyruvate kinase (PK, F) in microglial lysates. G) 3D confocal imaging of TOM20⁺ mitochondria and LysoTracker⁺ lysosomes showing mitochondrial‐lysosomal interactions. H) Western blot and quantification of iNOS, COX‐2, and TNF‐α in BV2 cells. I) Flow cytometry plots and quantification of EdU⁺ proliferating BV2 cells. J) Western blot analysis of MeCP2 K210 lactylation in BV2 cells. K) Extracellular acidification rate (ECAR) tracing in BV2 cells following Rot/AA (inhibit mitochondrial respiration) and 2‐DG (inhibit glycolysis) treatment. L,M) Quantification of basal glycolysis L) and glycolytic reserve (post‐2‐DG acidification, M) from ECAR assays. n = 3 or 4 per group. Data are presented as mean ± SD. Statistical analysis was performed using one‐way A–F,H,I,L,M) or two‐way ANOVA K) with Tukey's post hoc test. ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Glucose uptake was measured using 2‐NBDG (Elabscience, E‐CK‐A441), a fluorescent glucose analog ( E x / E m = 475/550 nm).

Techniques: Activation Assay, Mutagenesis, Flow Cytometry, Expressing, Plasmid Preparation, Staining, Membrane, Fluorescence, Activity Assay, Imaging, Western Blot

Journal: Advanced Science

Article Title: Flipping the Switch: MeCP2‐Mediated Lactylation Rewires Microglial Metabolism and Inflammation via the HK2/mTOR Axis in Poststroke Neuroinflammation

doi: 10.1002/advs.202513400

Figure Lengend Snippet: MeCP2–HK2 axis mediates mitochondrial dysregulation and proinflammatory metabolism in microglia under inflammatory stress. A,B) Representative whole‐brain A) and cortical B) immunofluorescence images showing elevated HK2 expression in Iba1⁺ microglia at 3 days post‐tMCAO; quantification of HK2 intensity in Iba1⁺ microglia (B, right). n ≥ 20 cells per group. C) Western blot showing increased HK2 protein levels in murine BV2 cells following LPS+IFN‐γ treatment. n = 3 per group. D) Primary microglia exhibit increased HK2 expression upon LPS+IFN‐γ stimulation; quantification of HK2 intensity in Iba1⁺ primary microglia (D, right). n ≥ 20 cells per group. E,F) HK2 overexpression (OE‐HK2) increases mitochondrial depolarization (MitoTracker), calcium influx (Fluo‐4), mitochondrial ROS (MitoSOX), and glucose uptake (2‐NBDG). G) OE‐HK2 decreases ATP content and pyruvate dehydrogenase (PDH) activity while increasing pyruvate kinase (PK) activity. n = 3 per group. H) Western blot shows that HK2 promotes iNOS, COX‐2, and TNF‐α expression. n = 3 per group. I,J) HK2 knockdown (si‐HK2) reverses mitochondrial depolarization, Ca 2 ⁺ overload, ROS generation, and glucose uptake. K) si‐HK2 increases ATP and PDH activity while suppressing PK activity. n = 3 per group. L) si‐HK2 significantly reduces iNOS, COX‐2, and TNF‐α levels in murine BV2 cells. n = 3 per group. Data from animal experiments are means ± SEM; those from cell line experiments are means ± SD. Statistical analysis was performed using t ‐test B–D,G,H,K,L). ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Glucose uptake was measured using 2‐NBDG (Elabscience, E‐CK‐A441), a fluorescent glucose analog ( E x / E m = 475/550 nm).

Techniques: Immunofluorescence, Expressing, Western Blot, Over Expression, Activity Assay, Knockdown

Journal: Cell Death & Disease

Article Title: FBP1 orchestrates keratinocyte proliferation/differentiation and suppresses psoriasis through metabolic control of histone acetylation

doi: 10.1038/s41419-024-06706-6

Figure Lengend Snippet: a Ctrl or shFBP1-2 HaCaT cells were incubated for an additional 24 h in growth medium 3 days after plating. Medium was collected and lactate content was measured, n = 3 independent experiments. b Ctrl or shFBP1-2 HaCaT cells were treated with 2-NBDG (100 μM) for 30 min and images were taken with an excitation wavelength of 485 nm and an emission wavelength of 535 nm. Scale bars: 100 µm. c Extracellular acidification rate (ECAR) of Ctrl and shFBP1-2 HaCaT cells were detected using a Seahorse XF extracellular flux instrument, n = 3 independent experiments. d Ctrl or shFBP1-2 HaCaT cells were treated with 0 mM (−), 1 mM (+) or 2 mM (++) 2-DG for 2 days, and then exposed to 0.06 mM (−) or 1.2 mM (+) calcium for 6 days before collection. The indicated proteins were detected by western blots. e Ctrl or shFBP1-2 HaCaT cells were plated (1000 cells/well) and cultured with or without 1 mM 2-DG for 2 weeks. The colonies were stained with 0.1% crystal violet and counted, n = 3 independent experiments. Data are shown as mean ± s.d. Statistical analyses in ( a ), ( c ) and ( e ) were performed with Student’s t -tests. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Glucose uptake in HaCaT cells was measured by analysis of 2-NBDG taken up by cells using 2-NBDG Glucose Uptake Assay Kit (BioVision, K682-50).

Techniques: Incubation, Western Blot, Cell Culture, Staining