Gelatine-coated plates

Imprint with gelatine-coated plates Imprint with thermoplastic films... 

There are two different widely used imprint methods in damage analysis, namely imprints on gelatine-coated plates and on thermoplastic films, usually polypropylene or polystyrene. In Table 8.7 the most important advantages of these complementary imprint methods are compared. 

Principle of the method with gelatine-coated plates... 

Once prepared, feeders can be kept in culture for up to 2 wk prior to use, but must remain as an intact monolayer. Most researchers prepare feeders on gelatin-coated plates. Although this is not essential for the propagation of the fibroblasts themselves, it promotes tight adherence to the substratum, and is also essential for the adherent growth of ES cells to any regions devoid of fibroblasts. 

A modified version of the ECVAM-approved embryonic stem cell test (EST) (52) was recently developed by Hunter and coworkers in NHEERL (31). This assay is capable of quantitatively assessing cytotoxicity and cardiomyocyte differentiation and was used to test the ToxCast Phase I chemical library (32). Briefly, male murine J1 mES cells were seeded onto gelatin-coated 96-well plates at a known density in differentiation media on day 0. On day 1, cells were treated with chemicals ranging from 0.0125 to 12.5 pM, and on day 9 In-Cell Western (Li-Cor Biosciences) assays were assayed for a- and P-cardiac Myosin Heavy Chain (MYH6/MYH7)... 

Repeat this procedure with each of the photographic plates. You may find that varying time intervals and different thicknesses of the gelatin coating produce more or less successful results. You will want to experiment a bit until you find the best time exposure for the plates you have made. 

Chick embryo muscle cell plating efficiency was low on albumin and y globulin coated surfaces but the fibrinogen and gelatin coated surfaces were strongly cell adherent. 

Plate in a gelatin-coated 80-cm tissue-culture flask (Nuclon) with 20 mL of growth medium. The latter is added to enrich the plated culture in transfected F-9 cells the transfected cells are geneticin-resistant, but untransfected F-9 cells are not. 

Preparation of FITC-Gelatin Coated Coverslips. Materials FITC-conjugated gelatin, 12-well glass coverslips, 12-well tissue culture plates, glutaraldehyde, PBS, sodium borohydride (Sigma, S-9125), and SF RPMI. 

Seed 10 LOX cells per labeled gelatin-coated covershp in a 12-well plate, 1-2 days after transfection. Generally, cells from each transfected well of a 6-weU tissue culture plate are trypsinized and re-seeded at a 1 6 to 1 8 dilution onto each coverslip in 1 ml complete RPMI. LOX cells are cultured for 16-24 hrs on gelatin coverslips for degradation/invasion assays. Experiments are repeated three times with two separate coverslips per experimental condition. 

Gelatin-coated culture plates or flasks Coat culture plates or flasks for 2 h with 0 1 % gelatin/PBS solution Aspirate off and wash once with PBS. Cells can be plated imraediumtely or, alternatively, the plates or flasks can be stored in PBS until needed... 

To prepare gelatin-coated 100-mm cell culture plates, add enough 0.1% gelatin solution to cover the surface of the culture vessel and leave at ambient temperature for 2-3 h. 

EB formation from intact colonies is much more efficient than from pieces of cut up colonies. To facilitate removal of colonies, we therefore switch Matrigel for a solution of 0.1 % gelatin and a low amount of Matrigel to coat plates, providing just enough attachment to maintain pluripotency. 

To verify that EB differentiation progressed normally from day 0 to 4 or to optimize day 1-4, a differentiation control can be used. Day 4 EB are plated on 0.1 % gelatin coated dishes in... 

A transition step between the reaction system just considered (silver ions, p-phenylenediamine, silver sol) and actual physical development of a fixed photographic plate or film is supplied by the investigations of Arens (27, 28). He used a series of silver, gold, and silver sulfide sols which had been coated on glass plates, using gelatin as the binding material. In this way he obtained plates in which colloidal particles... 

The silver halide crystals can be formed as microscopic grains suspended in a protective colloid, usually gelatin. This dispersion, the photographic emulsion, can be coated on a suitable support to obtain photographic films, plates, and papers. 

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