Embedding cells agarose

The ReProComet assay (repair-proficient comet assay) was developed to detect chemically induced DNA damage in sperm cells. In order to overcome the intrinsic DNA repair deficiency of the sperm cells this modified comet assay is based on the addition of a protein extract from HeLa cells to agarose-embedded sperm on microscopic slides previously exposed to mutagenic compounds. A clear-cut dose-dependent effect was measured after addition of the cell extract representing a proof of concept of a novel in vitro mutagenicity test on sperm [18]. Transferability to other laboratories remains to be addressed. 

Embedding cells in agarose on microscope slides 427 Staining embedded DNA 428... 

For illustrative purposes, the method used by Morgan4 is described below. In this example, cells are re-suspended in agarose gel that is taken up in a 1 mL syringe (see Fig. 6.4), generating a cylinder of cells. The cylinder of cells can be treated like tissue and placed in wax which, in turn, can be cut and embedded in paraffin wax blocks for cutting on a microtome. 

Notes HeLa cells (1 x 106) were formalin-fixed in an equal volume of 1% agarose. After histological processing and paraffin embedding, the cell plugs were rehydrated and resuspended in the indicated buffer. Total protein in the supernatants was assessed colorimetrically after heating at the indicated temperatures and times. The % recovery values are the mean, the standard deviation and relative to a fresh cell lysate from the sample number of cells (for more detail, see Reference 25). 

Figure 4.1 The comet assay. A single-cell suspension is embedded in agarose on a slide. Cells are then subject to lysis followed by electrophoresis. If present, damaged DNA migrates out of the nucleoid structure during electrophoresis to producing a characteristic comet shape. Double-strand breaks are revealed under neutral conditions, whereas alkali conditions additionally show single-strand breaks and alkali labile sites. Image analysis of stained DNA is used to quantitate the amount of damaged DNA in the comet tail.

Most species of red algae (Rhodophyta) have a cell wall with an inner cellulose layer embedded in a matrix of agarose and agaropectin mucilage. This embedded... 

Moskaluk, C. A. and M. H. Stoler. 2002. Agarose mold embedding of cultured cells for tissue microarrays. Diagn Mol Pathol 11 234-8. 

Transmission electron microscopy (TEM) is important to monitor cell-free sperm decondensation and nuclear formation. To prepare specimens for TEM, incubation mixture aliquots were fixed for 45 min in 2.5% (v/v) paraformaldehyde, 3.1% (v/v) glutaraldehyde, 0.02% (w/v) picric acid in 30 mAf NaHP04, pH 7.5. After fixation, samples were embedded in 2% (w/v) low-gelling-temperature agarose and postfixed for 15 min in 1% (w/v) OSO4. Samples may be dehydrated either in ethanol and propylene oxide or acetone and embedded for sectioning in Spurr s low-viscosity epoxy resin (Spurr, 1%9). Before examination, sections of about 70-nm thickness should be stained, e.g., with uranyl acetate and Reynolds lead citrate (Re)molds, 1%3). 

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