Evos xl core microscope

HUVEC spheroid outgrowth assays were performed as described previously.⁶⁹ (link) Spheroids were stimulated in EBM (6% FCS) containing 50 ng/mL VEGF-A 165 for 16 h before the addition of 4% PFA to the medium. Images of 10 spheroids per condition and replicate were acquired using an Evos XL Core microscope (Life technologies) and outgrowth length and numbers quantified using ImageJ.

GBM cell lines were seeded at densities of 300,000 cells per well in a 6-well plate and transfected 24 h later with either miR-3928 or scrambled control. After 72 h, the cells were seeded with at a density of 417 cells/mL in 0.01% FBS media in collagen IV coated chambers. The chambers were incubated in complete media with 10% FBS in a 24-well plate in triplicates. After 8 h of incubation at 37 °C in 5% CO₂ and 20% O₂, the chambers were gently rinsed with 1xPBS and stained and fixed with 0.1% crystal violet solution in 20% methanol. After drying at room temperature, the chambers were imaged on Evos XL Core microscope (Life Technologies, Grand Island, NY, USA). The images were analyzed with ImageJ Software (National Institutes of Health, Bethesda, MD, USA).

E. coli was cultured at 37 °C under aerobic conditions with shaking in Luria Bertani (LB) medium overnight. The bacteria were added to macrophage cultures at a ratio of 1:2500 (v/v) when the bacterial culture reached an OD of 0.6 as determined by spectrophotometry, followed by incubation for 1 h at 37 °C. Subsequently, cell medium or cell lysates were collected for the colony formation experiment. Image capture was conducted using an EVOS XL Core Microscope (Life Technologies).
LB medium was prepared by dissolving 1 g tryptone, 0.5 g yeast extract, 1 g NaCl, and 1.4 g agar powder in 100 ml distilled water, followed by sterilization at 121 °C for 25 min. LB medium was then poured into petri dishes in an ultra-clean workbench and allowed to cool and solidify.
Equal volumes of BMDM medium or cell lysates from peritoneal lavage fluid of each group were added onto the solid LB medium and spread evenly with a sterile spreader until dry. The amount of liquid added to the plate was adjusted according to the number of colonies in the culture medium. The coated LB plates were sealed with sealant and incubated at 37 °C overnight for 24 h to observe the number of colonies.

Each cell sample was assayed in triplicate. Cells were counted with a TC 20 Automated Cell Counter (Bio-Rad Laboratories, Inc., Hercules, CA, USA). A specific number of cells (for HN5, 5 × 10³ cells/well; for Hs-680Tg, 8 × 10³ cells/well; and for PC3, 1 × 10⁴ cells/well, all cells in 100 μL culture medium) were plated in clear 96-well cell-culture plates overnight and then cells were treated with the greatest clinical exposure concentration (i.e., if 120 markers [the maximum number likely to be used in one patient with a prostate volume of 60 cc] were to leak simultaneously into the human periprostatic area after implantation) of C4 or its components (1% CoCl₂·6H₂O, or the 2% NAC, or the Co : NAC [1% : 2%]), and cells were incubated at 37°C. At 90 minutes after treatment, pictures of cells were taken with a bright field Evos XL core microscope (AMEX 1000, Life Technologies, Carlsbad, CA, USA) at ×10 magnification.

Adipogenesis was quantified by positive staining of lipid droplets with Oil Red O (Sigma) and osteogenesis by alkaline phosphatase activity (Sigma, following the manufacturer’s protocol). Adipogenic cells were imaged using an EVOS XL Core microscope (Life-Tech) with a Plan PH2 achromatic infinity-corrected 20 × lens.