Cell culture dishes

PAMAM dendrimers were synthesized starting from ethylenediamine according to a modified protocol described by Tomalia [31 (link)]. All reagents for chemical syntheses, i.e., glycidol (mixture of enantiomers), methyl acrylate, ethylenediamine, and solvents, were purchased from Sigma-Aldrich (St Louis, Missouri, USA) as reagent grade and used as received. For biological studies, Eagle’s Minimum Essential Medium (EMEM), Dulbecco’s Modified Eagle’s Medium (DMEM and DMEM: F-12), fetal bovine serum (FBS), penicillin and streptomycin solution were obtained from ATCC (Manassas, VA, USA). Trypsin-EDTA solution, phosphate-buffered saline (PBS) with and without magnesium and calcium ions, 0.4% trypan blue solution, fluorescent marker DAPI (4′,6-diamidino-2-phenylindole, dihydrochloride) were purchased from Thermo Fischer Scientific (Waltham, Massachusetts, USA). Hydrocortisone, 0.33% neutral red solution (3-amino-6-dimethylamino-2-methyl-phenazine hydrochloride), was obtained from Sigma-Aldrich (St Louis, Missouri, USA). Cell culture dishes were from Corning Incorporated (Corning, NY, USA) or Nunc (Roskilde, Denmark).

Cells were seeded into 35 mm × 10 mm cell culture dishes (Corning) and allowed to grow until confluency. Initiation of migration was achieved by scratching confluent cells with a p10 pipette tip. Motility was measured on a Ziess Axiovert 200M microscope mounted with a Perkin Elmer Ultraview ERS enclosed in a heated chamber (37°C) with 5% CO₂ injection. Images were acquired every 10 min for at least 15 h. After image acquisition, Volocity software was used to determine percent wound closure at designated time points and to analyze single cell tracks over time. Cell tracking data included average velocity and meandering index (calculated for each track by measuring the displacement of the cell from origin at time of observation and dividing by the track length), and t-test was used to determine significance.

Gold (III) chloride hydrate (99.999% trace metals basis), sodium citrate, red blood cell lysing buffer, and acid phosphatase, leukocyte acid phosphatase (TRAP) kits were purchased from Sigma-Aldrich (St Louls, MO). TRACP&ALP Assay kit was purchased from Takara (Seoul, Korea). Alendronate sodium trihydrate was purchased from TCI (Tokyo Chemical Industry Co., LTD, Japan). Macrophage colony stimulating factor (M-CSF) and recombinant murine sRANK ligand were purchased from Peprotech Korea (Seoul, Korea). RIPA lysis buffer and PBS 10X were purchased from Millipore. Protease inhibitor cocktail tablets were purchased from Roche Diagnostics Indianapolis (USA). ICR six-week old male mice, used for extraction of BMMs, were purchased from Young bio (Sung-nam, Korea). Minimum Essential Media (MEM) Alpha Medium, fetal bovine serum (FBS), antibiotic agents (penicillin/streptomycin, PS) and Dulbecco’s Phosphate Buffered Saline (DPBS) were purchased from GIBCO BRL (Invitrogen Co., USA). 48-well cell culture plates and cell culture dishes (100 mm × 20 mm) were purchased from Corning Incorporated (New York, USA). EZ-Cytox (enhanced cell viability assay kit) was purchased from Dogen (Seoul, Korea).

The NIH/3T3 cell line (American Type Culture Collection [ATCC], CRL-1658™, Manassas, VA, USA, RRID: CVCL_0594), a mouse fibroblast cell line, was cultured in Dulbecco modified Eagle medium (DMEM; Gibco®, Cat. #11962–092, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; qualified, Gibco®, Cat. #6140–079,) and 1% antibiotic-antimycotic solution (Anti-anti; Gibco®, Cat. #15240–062). The Neuro-2A (N2A) cell line (ATCC, CCL-131™, RRID: CVCL_0470), derived from a spontaneous neuroblastoma tumor of a strain A albino mouse, and the U251 cell line (Sigma-Aldrich, Cat. #09063001, St. Louis, MO, USA, RRID: CVCL_0021), derived from a malignant glioblastoma tumor, were cultured separately in Minimum Essential Medium α (MEM α, Gibco®, Cat. #12571–063) supplemented with 10% heat-inactivated FBS (Gibco®, Cat. #6140–079), 1% of nonessential amino acid (Gibco®, Cat. #11140–050), and 1% Anti-anti (Gibco®, Cat. #15240–062). All cell lines were seeded onto cell culture dishes (Corning, New York, NY, USA) and maintained in a 37°C humidified incubator with 5% CO₂. Medium was refreshed every 2~3 days.

Mouse embryos were electroporated at embryonic day 15 (E15), and acute coronal brain slices (240 μm) were prepared at E17 and E18. Occipital slices were transferred onto slice culture inserts (Millicell) in cell culture dishes (35 × 10 mm; Corning) with Neurobasal medium (Invitrogen) containing the following: B27 (1%), glutamine (1%), penicillin/streptomycin (1%), horse serum (5%), and N2 (1%). Slices were used for imaging (1–2 h after slicing) or for pharmacological treatments (incubated at 37°C in 5% CO₂, for 1 day). A subset of slices was incubated with the medium containing rapamycin (100 μM).

HeLa cell line of human cervical cancer was provided by the Xiamen Life Internet Technology Co., Ltd. BRD4 inhibitor JQ1 was purchased from MCE (MedChem Express). Before use, JQ1 was diluted and dissolved with dimethyl sulfoxide (DMSO) to adjust the storage solubility of JQ1 to 50 mmol/L. DMEM medium (containing high glucose with L-glutamine; with sodium pyruvate) was purchased from HyClone (Logan, UT, USA). Fetal bovine serum was purchased from PEAK, 0.25% Trypsin was purchased from Gibco and the CCK-8 kit (cell counting kit-8) was purchased from Biosharp. The reverse transcription kit was purchased from Xiamen Life Internet Technology Co. (Xiamen, China), cell culture dishes from Corning, and cell culture incubators from Forma Scientific (Thermo Fisher Scientific, Waltham, MA, USA). The experimental instruments included enzyme-linked immunoassay (Enzyme Analyzer; Bio-Rad, Herclues, CA, USA), Applied Biosystems 7500 Real-Time PCR system (Applied Biosystems, Waltham, MA, USA) and inverted phase contrast microscope (Nikon, Tokyo, Japan), etc.

The assay was adopted from the in vitro scratch assay protocol [10 (link)]. Patient's specimens from all time points were evaluated in a single experiment including a negative control. SW480 cells were cultured in cell culture dishes (6 mm × 15 mm, Corning, NY) and left overnight to form a confluent monolayer. The monolayer was scored to leave a scratch 0.5 mm wide, rinsed with phosphate buffered saline (Biological Industries, Israel), and replaced with fresh, serum-free culture medium containing 20% peritoneal fluid (this concentration was selected as optimal exposure not compromising cell viability, following calibration experiments using 0–100% peritoneal fluids). Negative control of this assay was evaluated using serum-free culture medium with L-glutamine, penicillin, and streptomycin supplementation only. Baseline level of the effect was determined using the presurgical peritoneal effusion or lavage fluids.
Digitized images of the plates were obtained at the start of the experiment (for dish baseline) and after 5 hours (×20, Olympus TH4-200 Microscope). Migration level was determined by the cells counted per scratch area (cells per pixel; Image-Pro Plus 6.0 Software, Media Cybernetics). Bar charts of migration levels were created using Microsoft Office Excel 2007.