Plasma membrane proteins were used as a positive control

hich differs from migration across plastic surfaces, we describe migration as the apparent movement of cells through porous hydrogels evidenced by extension of cellular processes we call “invadopodia”and by the merging of formerly separated clusters in the hydrogels. Materials and Methods Materials Electrophoresis, cell culture, and transfection reagents and supplies were purchased from Invitrogen. TCMTM defined serum replacement was purchased from MP Biomedicals. Hyaluronic acid was generously donated by Genzyme Corporation. RHAMM and CD44 antibodies were purchased from Novus Biologicals. Hyal2 and b-actin antibody were purchased from Abcam. Equine-a-mouse-HRP secondary antibody was purchased from Cell Signaling Technology. Hyal1 antibody, bovine testicular hyaluronidase, b-mercaptoethanol, glycine, TweenH20, bovine serum albumin, sodium formate, Coomassie brilliant blue, disodium cromoglycate, and deoxycholic acid were purchased from Sigma Aldrich. Goat-a-rabbit-HRP secondary antibody, protease inhibitors, 5X sample buffer, sulfo-NHS-SS-biotin, and avidin agarose were purchased from Thermo Scientific. Laemmli sample buffer and alcian blue were purchased from Biorad. Tris buffer, glacial acetic acid, methanol, low melting point agar, dimethyl sulfoxide, sodium dodecyl sulfate, Triton X-100, and sodium chloride were purchased from Fisher Scientific. Nonidet P-40 was purchased from Roche and ethanol was purchased from Decon Labs, Inc. All compounds used were reagent grade or better. Cell Culture Low passage number PCa cells were maintained in Corning tissue culture 75 mm flasks at 37uC in 5.0% CO2 in T-medium supplemented with 5% fetal bovine serum and 100 U/ml penicillin G sodium and 100 mg/ml streptomycin sulfate in 0.085% saline. Medium was changed every 23 days. Cells were passaged at approximately 80% confluency, judged by eye, using 0.25% trypsin with ethylenediaminetetraacetic acid 4NNa. LNCaP sublines were generously provided by Dr. Leland Chung and PC3 cells were purchased from ATCC. Preparation of HAALD and HAADH To synthesize the HA aldehyde, oxidation reaction of HA was performed in the presence of sodium periodate under aqueous conditions. Adipic dihydrazide -modified HA was synthesized by carbodiimide-mediated coupling of ADH with the carboxyl groups of HA in aqueous conditions. Detailed procedures for the synthesis and characterization of both of these HA derivatives was reported in our previous study. Cell Culture in 2D and 3D Conditions HAALD and HAADH were dissolved separately in Dulbecco’s phosphate buffered saline to a concentration of 10 mg/ mL LY3039478 web overnight at 37uC. 2% LMP agar in DPBS was prepared and was melted in a 70uC heatblock at least 1 hour prior to use. Dissolved HA derivates were sterilized by UV irradiation at 254 nm for 15 minutes prior to use. PCa cells were released from their flask using 0.25% Trypsin EDTA 4NA and the total number of cells was counted using a hemocytometer. 100,000 or 600,000 cells were pelleted for each culture. Cell culture inserts were pre-wet in T-medium then placed in the wells of a 24-well or 6-well culture plate. For 2D cultures, cell pellet was mixed with T-medium and plated. For 3D HA hydrogel culture, cell pellet was mixed with 100 ml or 300 ml of HAALD. An equal volume of HAADH was added and the culture was mixed well to evenly disperse cells. The hydrogel solution then was pipetted into pre-wet cell culture inserts and allowed to solidify for approximately 10 minutes at 37uC.