Journal: Current protocols

Article Title: Redefining Cell Culture Using a 3D Flipwell Co-culture System: A Mimetic for Gut Architecture and Dynamics In Vitro.

doi: 10.1002/cpz1.70107

Figure Lengend Snippet: Figure 15 THP-1 dramatic polarization toward an M1-like phenotype with Sepiapterin treatment in the 3D Flipwell coculture system.(A) Confocal images of THP-1 cells cocultured with Caco-2:HT- 29 in the 3D Flipwells and stained for pan macrophage marker CD68 (red) and CD80 (green). Pan macrophage CD68 marker is present in both the SEP treated sample and the untreated control. CD80 marker, typical of the M1 polarization phenotype, is more prominently present in the SEP treated sample with long pseudopodia extending outward typical of the M1 phenotype. (B) Con- focal images of THP-1 cells cocultured with Caco-2:HT-29 in the 3D Flipwell and stained for pan macrophage marker CD68 and CD163 to validate phenotypic polarization toward M1 phenotype and away from M2 phenotype. Scale bar = 25 μm.

Article Snippet: See the T erm s and C onditions (https://onlinelibrary.w iley.com s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense Anti-human MUC2 (996/1) (Thermo Fisher, cat. no. MA5-12345) Anti-human CK20 (Life Technologies, cat. no. PA5-82875) Anti-human CD80 (Cell Signaling Technologies, E3Q9V, cat. no. 15416) Anti-human CD163 (Abcam, cat. no. 156769) or anti-human CD68 (Novus Biologicals, cat. no. NB100-683) Secondary antibodies: Alexa Fluor 488 goat anti-rabbit IgG (Thermo Fisher, cat. no. A11008) for CD80 and CK20 Alexa Fluor 594 goat anti-mouse IgG (Thermo Fisher, cat. no. A11005) for MUC2 and CD68 or CD163 DAPI, VectaShield mounting medium (Vector Laboratories, cat. no. H-1000) 50-ml conical tubes (SpectraTube, Alkali Scientific cat. no. C2750) Plastic wrap Lab tape or shipping tape Tweezers 12-well tissue culture plate (Corning Costar, cat. no. 3513) 6-well dish Aluminum foil Scalpel Round cover slips, #1, 1 oz (Corning Float Glass, cat. no. 50143822, or Fisherbrand cat. no. 12-546-2 25CIR-2) Laboratory marker (VWR, cat. no 95042-566) Attofluor cell chamber coverslip holder (Thermo Fisher Scientific, cat. no. A7816) Confocal microscope and software [e.g., Leica Microsystems TCS SP5, multi-photon laser scanning, and Suite Advanced Fluorescence (LAS AF) software] Deep Petri dish bottoms, 100 × 25–mm deep polystyrene stackable Petri dish, sterile (USA Scientific, cat. no. 8609-0625) 10-, 200-, and 1000-μl pipet tips (Alkali Scientific PurePoint or equivalent) Beckman Coulter Allegra 6 centrifuge with a swing bucket rotor for 50-ml conical tubes Optional: ImmunoPen (Fisher Scientific, cat. no. 4021761EA) Wash, permeabilization, disassembly, and blocking steps 1.

Techniques: Staining, Marker, Control

Journal: Chronic Diseases and Translational Medicine

Article Title: C-X3-C motif chemokine ligand 1/receptor 1 regulates the M1 polarization and chemotaxis of macrophages after hypoxia/reoxygenation injury

doi: 10.1016/j.cdtm.2021.05.001

Figure Lengend Snippet: Down-regulation of CX3CR1 with siRNA attenuated the M1 polarization of macrophages induced by hypoxia/reoxygenation. (A and B) Representative Western blots and summarized data showing the protein levels of CX3CR1, TNF-α, and ARG-1 in different groups of RAW264.7 macrophages. (C) The expression analysis of CD80 and CD206 by flow cytometry in different groups of RAW264.7 macrophages. a P < 0.05, b P < 0.01 vs Control; c P < 0.05, d P < 0.01 vs H/R; e P < 0.05, f P < 0.01 vs CX3CL1 + H/R. H/R: hypoxia/reoxygenation. Cells were exposed to hypoxia condition for 6 h, then reoxygenation for 24 h. The concentration of CX3CL1 recombinant protein used was 50 ng/mL.

Article Snippet: The other antibodies used were as follows: anti-CD80 (bs-2211R, Bioss, Beijing, China), and anti-phosphorylated-NF-κB p65 (#3033, Cell Signaling Technology, Danvers, MA).

Techniques: Western Blot, Expressing, Flow Cytometry, Concentration Assay, Recombinant