Journal: STAR Protocols

Article Title: Protocol for isolating stromal cells from lymphoid tissue for performing scRNA-seq

doi: 10.1016/j.xpro.2026.104501

Figure Lengend Snippet: Cell selection using automated magnetic cell sorting (A) Cells were stained with CD45-biotin and CD31-biotin and sorted using autoMACS® Pro Separator. (B) Number of cells before staining for autoMACS® separation (step 11), and number of cells recovered from positive selection (CD45 + and CD31 + cells) and negative selection (CD45 - and CD31 - cells) in step 23. Each dot represents combined numbers from inguinal, axillary and brachial lymph nodes from 6 mice. Colors indicate biological replicates. (C) Percentages of cells after separation compared to the pre-staining cell count performed in step 11. (D) Purity check of separated cells using flow cytometry and staining for CD45 and CD31. (E) Overlay plots of positive selection (blue) and negative selection (red). (F) Percentage of CD45 + , CD45 - and CD31 + cells recovered (∗∗∗∗ p value < 0.0001, ∗ p value < 0.05). (G) Final viability check of positive and negative selected cells acquired just before performing scRNA-sequencing analysis.

Article Snippet: CD31 antibody, anti-mouse, Biotin (Dilutions in 1:50) , Miltenyi Biotec , Cat# 130-119-562, RRID: AB_2751728.

Techniques: Selection, FACS, Staining, Cell Characterization, Flow Cytometry, Sequencing

Journal: The Journal of General Physiology

Article Title: Beat-locked ATP microdomains in the sinoatrial node map a Ca 2+ -timed energetic hierarchy and regional pacemaker roles

doi: 10.1085/jgp.202513874

Figure Lengend Snippet: Superior SA node myocytes exhibit elevated diastolic ATP and metabolic flux compared with the inferior region. (A) 3D segmented maximum-intensity projection of a whole-mount SA node immunolabeled for CD31 (vasculature, red) and cyto-iATP (myocytes, green). The dashed line denotes the boundary between superior and inferior regions. (B) Image-processing workflow illustrating merged maximum-intensity projections, binary segmentation masks, and extraction of grayscale cyto-iATP signals used for quantitative analysis. (C) Mean cyto-iATP fluorescence intensity per myocyte, grouped by region ( N = 5 mice per region), reporting expression levels of the EGFP-tagged cyto-iATP sensor. (D) Live confocal imaging of cyto-iATP signals showing representative line-scan images and corresponding normalized fluorescence traces (F/F 0 ) from superior and inferior regions. (E and F) Summary quantification of cyto-iATP signal mass rate (E) and estimated diastolic [ATP] i (F). P values are shown above comparisons. Large circles denote per-animal means; small circles indicate individual biological replicates. N represents the number of independent mice.

Article Snippet: For immunolabeling, SA nodes were incubated for 48 h at 4°C with a goat anti-mouse CD31 primary antibody (1:50, AF3628; R&D Systems).

Techniques: Immunolabeling, Extraction, Fluorescence, Expressing, Imaging

Journal: Molecular Metabolism

Article Title: Endothelial ADGRF5(GPR116) governs vascular adaptation required for sustained thermogenic remodeling of brown adipose tissue

doi: 10.1016/j.molmet.2026.102346

Figure Lengend Snippet: Endothelial reprogramming in BAT and iWAT upon ADGRF5(GPR116) deletion. (A) UMAP representation of vascular cell populations from BAT of WT and ADGRF5(GPR116)KO mice housed at 22 °C or 8 °C (14 days), based on integrated single-nucleus RNA-seq analysis. (B) Dot plot showing relative proportions of endothelial subpopulations in BAT across genotypes and temperatures. Dot size indicates the percentage of cells per condition. (C) UMAP representation of vascular and perivascular cell populations from iWAT of WT and ADGRF5(GPR116)KO mice at 22 °C and 8 °C (14 days), highlighting depot-specific differences in endothelial organization compared with BAT. (D) Dot plot showing relative proportions of major endothelial subtypes in iWAT across genotypes and temperatures. Nuclei numbers for each condition are indicated. (E) Module score analysis of endothelial functional programs in BAT (left) and iWAT (right), comparing WT and KO conditions. (F) Heatmaps showing transcription factor regulon activity in BAT (top) and iWAT (bottom) endothelial subtypes across genotypes and temperatures. (G) Representative immunofluorescence images of BAT sections stained for CD31 (endothelial cells) and Hoechst in WT and ADGRF5(GPR116)KO mice under cold exposure. (H) Quantification of CD31-positive area normalized to total region of interest (ROI) area, in BAT, iWAT, and gonadal WAT (gWAT), (I) qPCR analysis of endothelial marker expression ( Pecam1 , Cdh5, Cldn5) in whole BAT tissue, from WT and ADGRF5(GPR116)KO mice, exposed to 8 °C for 14 days. Dots indicate individual mice. (J) Whole-mount CD31 immunostaining of iWAT vasculature from WT and ADGRF5(GPR116)KO mice following cold exposure, illustrating vascular network organization. (K) Quantitative analysis of vascular morphology parameters. Each symbol represents a different mouse. (L) FITC–dextran permeability assay showing endothelial barrier function in BAT, iWAT, gWAT, liver, kidney, and lung of WT and ADGRF5(GPR116)KO mice. (M) Representative Sirius Red/Fast Green–stained sections of BAT from WT and ADGRF5(GPR116)KO mice at 22 °C and 8 °C (14days). (N) Quantification of hydroxyproline content in BAT and iWAT, indicating total collagen content across genotypes and temperatures. Data are shown as mean ± SEM. Statistical analyses were performed using unpaired two-tailed Student's t test, Statistical significance is indicated as ∗ p < 0.05, ∗∗p < 0.01, otherwise, differences are not significant (ns).

Article Snippet: CD31 + cells were labeled with CD31 microbeads (Miltenyi, #130-097-418) and isolated using LS columns.

Techniques: RNA Sequencing, Functional Assay, Activity Assay, Immunofluorescence, Staining, Marker, Expressing, Immunostaining, FITC-Dextran Permeability Assay, Two Tailed Test

Journal: Molecular Metabolism

Article Title: Endothelial ADGRF5(GPR116) governs vascular adaptation required for sustained thermogenic remodeling of brown adipose tissue

doi: 10.1016/j.molmet.2026.102346

Figure Lengend Snippet: Endothelial ADGRF5(GPR116) is required to sustain thermogenesis during prolonged cold exposure. (A) Experimental schematic of prolonged cold exposure in tamoxifen-inducible EC-ADGRF5(GPR116)KO mice. Tamoxifen was administered prior to cold exposure to induce endothelial recombination, followed by a second tamoxifen injection after 7 days, 8 °C, to ensure continued endothelial ADGRF5(GPR116) deletion during the maintenance phase of cold adaptation. (B) qPCR analysis of Adgrf5(Gpr116) mRNA levels in CD31 + endothelial cells isolated from BAT of WT and endothelial inducible KO mice at 22 °C and after 7 days, 8 °C. (C) qPCR analysis of endothelial markers ( Pecam1 , Cdh5, Gpr116 ) and stromal marker ( Pdgfra ) in CD31 + endothelial fractions and flow-through (FT) fractions, demonstrating endothelial specificity of Adgrf5(Gpr116) expression and recombination. Data points in (B,C) represent individual mice. (D) Time-resolved energy expenditure during the first week (7 days), 8 °C. (E–J) Metabolic cage analyses, for the last 3 days of week 1 (7 days), 8 °C. (K) Time-resolved energy expenditure during the second week (14 day), at 8 °C, following tamoxifen re-injection. (L–Q) Metabolic cage analyses, for the last 3 days of week 2 (14 days), 8 °C. (R) Body weight, (S) lean mass and (T) fat mass, (U) organ weights of control and EC-Adgrf5(Gpr116) KO mice at the end 14 days, at 8 °C protocol. Mice were females, n = 3 per group, 10–12 weeks old. All mice were single-housed. Grey shaded areas indicate the dark phase. Statistical analyses for indirect calorimetry data were performed using CalR, with group differences assessed using general linear models, with or without ANCOVA, where appropriate to account for body weight or lean mass differences, as implemented in the CalR platform. Body composition parameters were analyzed using unpaired two-tailed Student's t tests. Data are presented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, otherwise, differences are not significant (ns).

Article Snippet: CD31 + cells were labeled with CD31 microbeads (Miltenyi, #130-097-418) and isolated using LS columns.

Techniques: Injection, Isolation, Marker, Expressing, Control, Two Tailed Test