Journal: Journal of visualized experiments : JoVE

Article Title: Digestion of the murine liver for flow cytometric analysis of lymphatic endothelial cells

doi: 10.3791/58621

Figure Lengend Snippet: A. Representative immunohistochemistry from a mouse liver stained with PDPN (blue/green) and F4/80 (brown). Lymphatic vessel (LV), blood vessel (BV) and macrophage (MΦ) are labeled. Scale bar is 100μm. Formalin Fixed paraffin embedded tissue was deparaffinized for 20 minutes in xylene. Hydrated to water through gradient of ethanol, and antigen retrieval was performed using perkin elmer pH6 antigen retrieval buffer in a pressure cooker for 15 minutes. Tissue was blocked using 0.1%BSA and stained using anti-mouse PDPN (8.1.1) 1:400 and anti-mouse F4/80 (MCA497) 1:100 for 1 hour at room temperature. Anti-hamster IgG HRP and anti-rabbit IgG HRP were used as secondary antibodies. DAB+ and Vina Green were used to detect the F4/80 and PDPN respectively. Tissue was counter stained with Hematoxylin and imaged on a Nikon eclipse Ti. B. As in A except PDPN in green, cytokeratin 7 in red and dapi in blue. Tissue was blocked using 5% donkey and 5% goat serum and stained using anti-mouse PDPN (8.1.1) 1:100 and anti-mouse cytokeratin 7 (ab181598) 1:200 for 1 hour at room temperature. Anti-hamster IgG AF647 and anti-rabbit IgG PE were used as secondary antibodies. Tissue was counter stained with DAPI and imaged. Scale bar is 100μm. Lymphatic vessel (LV), blood vessel (BV) and bile duct (BD) are labeled. C. Log fold change in Vegfr3 and prox-1 expression from sorted liver LECs based on staining in protocol (CD45-, CD31+, CD146lo/neg and PDPN+) compared to RAW cells or primary murine lymph node LECs. Sorted cells were passed through a QIAshredder column and RNA was extracted using a QIAgen RNeasy micro kit and cDNA was made using the Qiagen QuantiTect reverse transcription kit. Transcript abundance was normalized to the housekeeping gene, Gapdh for every sample. Primers for Vegfr3, prox-1 and Gapdh were obtained from Qiagen.

Article Snippet: Sorted cells were passed through a QIAshredder column and RNA was extracted using a QIAgen RNeasy micro kit and cDNA was made using the Qiagen QuantiTect reverse transcription kit.

Techniques: Immunohistochemistry, Staining, Labeling, Formalin-fixed Paraffin-Embedded, Expressing

Journal: Stem Cell Research & Therapy

Article Title: Immune response caused by M1 macrophages elicits atrial fibrillation-like phenotypes in coculture model with isogenic hiPSC-derived cardiomyocytes

doi: 10.1186/s13287-024-03814-0

Figure Lengend Snippet: aCM + M1 coculture resulted in higher occurrence of arrhythmias and electrophysiological changes. A Immunofluorescence (IF) image of NC-030 coculture of aCM and M1 (d10) stained for cTnT, CX3CR1, CD68 and DAPI, showing M1 connected to aCM and expressing activation and tissue-resident markers; NC-059 coculture stained for cTnT, CD68 and DAPI (scale bars 50µm). B IF images of NC-030 aCM and M1 cocultures (d8) stained for cTnT, Cx43, CD14 and DAPI show CD14 + macrophage expressing Cx43 while in contact with aCM (scale bar 50µm). C Schematic of experimental schedule and conditions tested in MEA assay. Conditions were: atrial cardiomyocytes only (aCM only), aCM + M0 macrophages (aCM + M0), aCM + M1 macrophages (aCM + M1), aCM + M1 conditioned medium (aCM + cond) and aCM only with activation factors added (aCM + act) D Exemplary MEA trace, showing electrogram (i.e., sodium spikes) over time with sudden beat rate change in a NC-030 aCM + M1 sample. E Scatter dot plots showing beat irregularity on d1 after activation. Mann Whitney test used to compare conditions. F Scatter dot plot of NC-030 one day before activation (d-1) and up to 3 days thereafter (d1-3), showing highly significant increase of irregularity in aCM + M1 after activation and loss of irregularity over time. G Bar graphs of NC-030 on d1 after activation comparing electrogram amplitude, beat rate, FPD and conduction velocity between conditions

Article Snippet: M1 cells in coculture suspensions were removed through CD14 magnetic bead cell sorting (CD14 Microbeads human, MS column, MiniMACS kit, all Miltenyi) according to manufacturer’s protocol. aCM cell populations were stored at -80°C and used for RNA extraction (as previously described).

Techniques: Immunofluorescence, Staining, Expressing, Activation Assay, MANN-WHITNEY