differentiation medium dm-2-l1  (ZenBio)

Journal: iScience

Article Title: Adipocyte-released adipomes in Chagas cardiomyopathy: Impact on cardiac metabolic and immune regulation

doi: 10.1016/j.isci.2024.109672

Figure Lengend Snippet: T. cruzi infection induces adipocyte apoptosis, releasing adipomes that regulate adipogenic signaling (A) Immunoblot analysis of cleaved Caspase 7 expression in the lysates of 3T3-L1 adipocytes (uninfected, TNFα-treated, and T. cruzi infected) ( n = 3/group). Bar graph values were derived from densitometry analysis and by normalizing target protein expression to β-Actin. (B) Fold change expression of CASP3 , RIPK3, and MLKL mRNA transcripts (normalized to HPRT ) in cultured human adipocytes (uninfected, TNFα-treated and T. cruzi infected) ( n = 3). (C and D) Adipome size distribution and absolute concentration as determined by Spectradyne nCS1 with a C-900 microfluidic cartridge. Both mouse and human adipomes were derived from cultured 3T3-L1 and human adipocyte-conditioned media, respectively. Inset: A (blue) = uninfected, B (green) = TNFα-treated, C (red) = T. cruzi infected. (E) PCR analysis of adipogenic ( Adipoq, Fabp4 , and Pparg ), apoptotic ( Chop ) and extracellular vesicle ( Tsg101 and Cd13 ) marker genes in 3T3-L1-derived adipomes. Lane 1, 2, and 3 represent 3T3-L1-derived adipomes from three independent cultures. Product size: Adipoq , 192bp; Fabp4 , 133bp; Pparg , 132bp; Chop , 118bp; Tsg101 , 103bp; and Cd13 , 121bp. (F and G) Fold change expression of Adipoq mRNA transcripts (normalized to Hprt ) in RAW macrophages (F) and human fibroblasts (G) treated with 3T3-L1- and human adipocyte-derived adipomes, respectively, at 1:1 and 1:50 cell-to-adipome ratio for 48 h ( n = 3). All treatment groups (Treated - A, - B and - C) were compared to untreated groups. The # symbol indicates comparison between Treated - B and - C. Treated - A = adipomes from uninfected adipocytes; Treated - B = adipomes from TNFα-treated adipocytes; and Treated - C = adipomes from T. cruzi -infected adipocytes. Data are represented as mean ± SEM. (∗/# p < 0.05, ∗∗ p ≤ 0.01, ∗∗∗/### p ≤ 0.001 and ∗∗∗∗ p ≤ 0.0001).

Article Snippet: 3T3-L1 Differentiation Medium , ZenBio , Cat#DM-2-L1.

Techniques: Infection, Western Blot, Expressing, Derivative Assay, Cell Culture, Concentration Assay, Marker, Comparison

Journal: iScience

Article Title: Adipocyte-released adipomes in Chagas cardiomyopathy: Impact on cardiac metabolic and immune regulation

doi: 10.1016/j.isci.2024.109672

Figure Lengend Snippet: Adiponectin enables selective capture of adipomes from murine white adipose tissue (WAT) (A) Immunoblot analysis of phospho-Perilipin, cleaved Caspase 7 and Annexin V expression in the WAT lysates of infected (20 DPI) and uninfected C57BL/6J mice (n = 4–8 per group). Bar graph values were derived from densitometry analysis and by normalizing target protein expression to GDI. Error bars indicate the standard error of the mean (∗ p < 0.05). (B) Immunoblot analysis of adiponectin in WAT-derived large-EVs (L-EV) and small-EVs (S-EV). Black arrow points to the 25 kDa marker band on the pre-stained protein ladder. Adiponectin band appears at 26/30 kDa. Lanes: C, control WAT lysate; L Ev, large-EV; and S Ev, small-EV. (C) Surface immunofluorescence staining of 3T3-L1 adipocytes show the co-localization of Adiponectin (green) with Annexin V (red) (top panel) and FABP4 (red) with Annexin V (green) (bottom panel) on budding apoptotic bodies. Scale bar, 50 μm. (D) Immunoblot analysis of adiponectin in WAT-derived large-adipomes and small-adipomes. Black arrow points to the 25 kDa marker band on pre-stained protein ladder. Adiponectin band appears at 26/30 kDa. Lanes: C, control WAT lysate; L, large-adipomes; and S, small-adipomes. (E) Immunofluorescence assay (IFA) of bead-bound adipomes stained with Adiponectin-AF488 (top panel) and FABP4-AF488 (bottom panel) imaged on the FITC channel along with their corresponding bright field (BF) images. Scale bar, 5 μm. (F) WAT-derived adipome size distribution and absolute concentration as determined by Spectradyne nCS1 with a C-2000 microfluidic cartridge. Inset: L (blue), large-adipomes; S (green), small-adipomes. Gate: G1, particle concentration at size range of 250–400 nm diameter showing more small-adipomes than large-adipomes; and G2, particle concentration at size range of 700–1100 nm diameter showing mostly large-adipome distribution. (G) Transmission Electron Microscopy (TEM) images of small-adipomes (left, scale bar 80 nm) and large-adipomes (right, scale bar 100 nm) with a direct magnification of 20,000×. Black arrow indicates the budding scars on L-adipomes.

Article Snippet: 3T3-L1 Differentiation Medium , ZenBio , Cat#DM-2-L1.

Techniques: Western Blot, Expressing, Infection, Derivative Assay, Marker, Staining, Control, Immunofluorescence, Concentration Assay, Transmission Assay, Electron Microscopy

Journal: iScience

Article Title: Adipocyte-released adipomes in Chagas cardiomyopathy: Impact on cardiac metabolic and immune regulation

doi: 10.1016/j.isci.2024.109672

Figure Lengend Snippet:

Article Snippet: 3T3-L1 Differentiation Medium , ZenBio , Cat#DM-2-L1.

Techniques: Infection, Recombinant, Electron Microscopy, SYBR Green Assay, Lysis, Protease Inhibitor, Plasmid Preparation, XF Assay, Isolation, Quantitation Assay, Bicinchoninic Acid Protein Assay, Software