Celltracker green cmfda

In vivo macrophage migration into epididymal fat pads of mice was performed as published earlier 4, 23. Ten‐week‐old male C57BL/6 wild‐type (WT) mice purchased from Charles River Laboratory (Wilmington, DE, USA) were used. All mice were fed with standard chow and water ad libitum and experiments were approved by Yale University's Institutional Animal Care and Use Committee (2015‐10756). Five mg/kg LPS (Sigma‐Aldrich, St. Louis, MO, USA) only, LPS together with either 20 μg mouse D‐DT or the equal amount of mouse MIF was injected into the epididymal fat pads via a small incision to induce local adipose tissue inflammation. Control mice were treated with the equal volume of PBS. Thioglycollate elicited peritoneal macrophages (PM) were labelled with Cell Tracker Green CMFDA (Cell Tracker Green CMFDA Dye; Life Technologies, Carlsbad, CA, USA). Next, labelled PMs were injected retro‐orbitally. Mice were killed after 48 hrs and epididymal fat tissue was harvested, and the stromal vascular fraction (SVF) was isolated. The isolated ATM were stained with Cd11b AlexaFluor700 (eBioscience, San Diego, CA, USA), F4/80 eFluor450 (eBioscience) and Cd45 APC (Biolegend, San Diego, CA, USA) as reported earlier 4.

Human prostate cancer cells, LAPC4, VCaP, DU145, PC-3, and LNCaP cells were obtained from the ATCC (Manassas, VA), which also provided authentication of all cell lines. The cells were grown in ATCC-recommended medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin. The authenticated human prostate carcinoma C4-2 was purchased from UroCor. Inc (Oklahoma City, OK) and grown in T medium containing 5% FBS. Cells from each of cell line were frozen at early passages and kept in liquid nitrogen providing a stock for cultured cells that were used within 6 month after thawing. Tetracycline responsive PC-3 cells were maintained in media recommended by ATCC for parental PC-3 cells supplemented with 10% Tet system-approved FBS from Clontech (Mountain View, CA). All culture media were purchased from Life Technologies (Carlsbad, CA); fetal bovine serum (FBS) was purchased from Sigma-Aldrich (St. Louis, MO). Doxycycline (Dox) was from Clontech. Collagen type I, fibronectin, and Matrigel were from Becton Dickinson Biosciences (Bedford, MA). CellTracker™ Green CMFDA was from Life Technologies. Bisindolylmaleimide I (BIM-I) was purchased from AdipoGen (San Diego, CA) and phorbol 12-myristate 13-acetate (PMA) was purchased from Sigma-Aldrich. AZD5363 and LY294002 were purchased from Selleckchem (Houston, TX).

The adhesion assays were performed as previously described [22 (link)]. Briefly, black 96-well plates (Corning Costar, Celbio, Milan, Italy) were coated with FN (10 μg/mL). Cells were counted, stained with CellTracker green CMFDA (12.5 μM, 30 min at 37 °C, Life Technologies), and after three washes with 1% BSA (bovine serum albumin) in HBSS (Life Technologies) were preincubated with increasing concentrations of the new peptide ligands (10⁻¹⁰–10⁻⁴ M) or with the vehicle (methanol) for 30 min at 37 °C. Afterwards, cells were plated (500,000/well) on coated wells and incubated for 30 min at 37 °C. After three washes, adhered cells were lysed with 0.5% Triton X-100 in PBS (30 min at 4 °C) and fluorescence was measured (Ex485 nm/Em535 nm) in an EnSpire Multimode Plate Reader (PerkinElmer, Waltham, MA, USA).
The number of adherent cells was obtained by comparison with a standard curve prepared in the same plate using diverse concentrations of cells. Experiments were carried out in quadruplicate and repeated at least three times. Data analysis and IC₅₀ values were calculated using GraphPad Prism 6.0 (GraphPad Software, San Diego, CA, USA).

Melanoma tri-cultures were generated according to Klicks et al. (2019) (link). Briefly, 1 × 10⁴ cells of CCD-1137SK cells were seeded, followed by simultaneous addition of HaCaT (1 × 10⁴ cells/well) and SK-MEL-28 (ATCC) (2.5 × 10³ cells/well) after three days. For discrimination of individual cell types, HaCaT and SK-MEL-28 cells were labeled with CellTracker Red CMPTX (10 μM) and CellTracker Green CMFDA (10 μM) dye (both Life Technologies), respectively according to manufacturer instructions for 30 min. Tri-culture spheroids were kept in culture for another two days to reach an average diameter of 300 μm.