Purchase Instant Access. View Preview. Learn more Check out. Abstract en Studies on in vitro proteolysis of casein using dissolved trypsin or covalently bound to oxirane beads have shown that immobilization leads to a change in the peptide pattern of the resulting proteolysate. Citing Literature. Volume 38 , Issue 6 Pages Related Information. Close Figure Viewer. Browse All Figures Return to Figure. Previous Figure Next Figure. Email or Customer ID. Forgot password? Old Password. New Password. Password Changed Successfully Your password has been changed.
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Request Username Can't sign in? Forgot your username? Enter your email address below and we will send you your username. Forgot your password? Each layer was produced separately in polydimethylsiloxane PDMS , using a soft lithography approach. The detailed description of the fabrication protocol was discussed elsewhere. Two inlets allow the flow of a single drug or double drug combinations into the device Fig. Numbers of PDMS replicas could then be fabricated easily. The fabrication of the primary mold for the rounded wells was more complex. Hence, we applied a diffuser back-side lithography procedure, 94 which works using SU-8 negative tone photo-resist.
Back-diffuser lithography works by exposing to UV-light a thick photo-resist coated on the surface of an optical mask from the back and through an optical diffuser, such as an opal diffuser plate. In this way, it is possible to define rounded volumes of exposed photo-resist. After post-baking and development, ellipsoidal pillars of hardened SU-8 remain on the mask plate and could be used as a primary mold for soft lithography. The number and distribution of these lenses were defined by the openings in the optical mask, therefore the choice of the number of channels and the density of wells for each channel can be freely defined.
Instead of using the optical plate directly as a primary mold, with the purpose of increasing its lifetime we choose to use a double-replica strategy. The first replica made in PDMS out of the optical plate was with ellipsoidal wells same polarity as in the final device , but a second replica out of this PDMS mold was with ellipsoidal pillars, same as in the original optical plate. This last PDMS replica was finally used as a working mold for the fabrication of multiple layers with wells for the final device application.
Blood cells and cell lines were controls for components of the clinical patient samples, mainly white blood cells and cancer cells respectively. A total of 2. Cells were seeded at a concentration of — cells per microwell for rapid establishment of cancer cell clusters. The media could be replaced manually or with a syringe pump. Microwells with denser cell packing reflect lower grey values, which will be detected as clusters. Grey values of two regions from each image were obtained: 1 Background non-microwell and 2 Cell region within microwell , using an image processing software ImageJ.
Doxorubicin Sigma, cat. D and Aspirin Sigma, cat. Working concentrations of drugs were freshly prepared before use. Drugs could be introduced after overnight incubation or once clusters were established. For evaluating IC50 values of monolayer cultures, cells were seeded at 0. For screening of cancer cell clusters under various drug combinations, drugs could be introduced manually or via the gradient generator component of the assay.
Loaded syringes were connected to the assays via designated inlets. Ethidium bromide was not used to avoid fluorescence emission overlap with doxorubicin. Alternatively, samples could be stained as single cells after gentle release from microwells by pipette resuspension. Images should be obtained with the appropriate filters to reduce background signals from doxorubicin. For immunostaining with other antibodies, clusters were released from the microwells and resuspended as single cells for analysis. For cytoplasmic proteins Caspase-3; , cat.
Samples were incubated in the antibody cocktail for an hour, washed with PBS and imaged with confocal microscopy. To establish the IC 50 values, z-stacks images of 25 microwells from each channel were obtained. Images from each stack were merged based on maximum intensity. Merged images were processed to identify maxima signals corresponding to each cell. For consistency, the microwells considered for evaluation were obtained at the same distance from the assay inlets.
Images were obtained with confocal microscopy and processed with the ImageJ processing software. Processed images were merged to locate regions with overlapping CD44 and CD24 signals i. Treated cultures were transferred as a single-cell suspension to a new microwell-based cluster assay to determine cluster-forming potential. Similarly, for spheroid assays, single cells were mixed to a final concentration of 0. Spheroids were stained with Hoechst and imaged with an Olympus inverted confocal microscope Olympus, Tokyo, Japan.
All assays were maintained for 2 weeks prior to analysis. Cultures were lysed and homogenised by freeze-thaw with dry ice and sonication in cold buffer. The plate was washed three times with washing buffer PBS with 0. A standard curve was established using the absorbance gotten from a serial dilution of recombinant IL-6 Absolute concentrations of the samples were calculated using Prism 7 Graphpad Software Inc.
The relative oxidative activity of cultures was measured with the MTT assay Roche and peroxidase assay. Cultures were maintained and treated for 72 as previously described, before being harvested by gentle agitation. Each sample was tested in duplicate with the average of eight readings per well recorded. Desired fractions were collected, frozen in liquid nitrogen and lyophilised using a VirTis benchtop freeze dryer. The Zorbax Eclipse Plus C18, 2.
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Data were recorded with Masshunter Acquisition B6. Chemical shifts ppm were recorded with a residual solvent peak as an internal reference. Cultures were lysed and homogenised with freeze-thaw and shear force cell disruption in buffer RLT as provided. All primer specificities were confirmed with a single peak during melting curve analyses. All measurements were performed in duplicate and mean values of relative expression are shown.
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All error bars represented standard deviation of triplicate cultures from different samples. Adjusted multivariate analyses for continuous independent variables to other variables required larger sample sizes and were not utilised in this study. Resultant viability percentages were normalised to that obtained from samples in the last channel lowest drug concentration. The data sets supporting the conclusions of this article are stored in a secured research database and may be available upon presentation of formal approval.
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In Memoriam of Aydin Sayili: Biography and Account of his Scientific Activity
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