Cell dissociation

Shown in Figure 6 is in vitro cytotoxic activity of PIPAAm-PBMA micelles loaded with ADR or micelles without ADR at 29°C (below the LCST) and at 37°C (above the LCST) compared with that of free ADR. In vitro cytotoxic activity was measured using bovine aorta endothelial cells. Bovine aortic endothelial cells were obtained as previously reported using dispase for cell dissociation from freshly harvested bovine aorta [13]. The cells plated at a density of 3 x cells/well, were exposed with free ADR or micelles loaded with ADR at below and above the LCST for 5 days. In order to assay cytotoxicity of the free ADR or micelles loaded with ADR, culture medium was replaced with 10% FBS-supplemented phenol red-free DMEM containing 10% alamar Blue, a dye that is subject to reduction by cytochrome c activity and changes the color from blue to red [38]. After 4-hour incubation, reduction of the dye was estimated by absorbance at 560 and 600 nm. PIPAAm-PBMA polymeric micelles loaded with ADR showed higher cytotoxic activity than that of free ADR at 37°C (above the LCST)... 

Remove the PBS and wash the cells similarly once with 1 ml cell dissociation buffer. 

Dissociate the cells to a single cell suspension by gently pipetting the cell dissociation buffer on top of the cells and by carefully pipetting the cells up and down in the dissociation buffer. 

Deactivate the cell dissociation buffer by adding 1 ml complete medium to the dish. 

Egelrud T, Lundstrom A. The dependence of detergent-induced cell dissociation in non-palmo-plantar stratum corneum on endogenous proteolysis. J Invest Dermatol 1990 95 456-459. 

Stratum Corneum Cell Dissociation Involves Proteolysis. 73... 

STRATUM CORNEUM CELL DISSOCIATION INVOLVES PROTEOLYSIS... 

Experimental evidence that protein structures are involved in stratum corneum cell cohesion was presented by Bisset et al.9 They induced cell dissociation in pig and human nonpalmo-plantar stratum corneum by means of incubation of the tissue in the presence of the zwitterionic surfactant 6-octadecyldimethyl ammoniohexanoate. Cell dissociation could not be induced when the tissue had been pretreated with the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF). The fact that cell dissociation was found only in the presence of EDTA suggested a role also for calcium in stratum corneum cell cohesion. 

Lundstrom and Egelrud10 found a unipolar spontaneous cell dissociation in pieces of hypertrophic human plantar stratum corneum incubated in a simple buffer. The cell dissociation occurred only at the surface that had faced outward in vivo. The rate of cell dissociation was increased in the presence of EDTA. It was inhibited by inhibitors of serine proteases, but not by inhibitors of other groups of proteases. Since the tissue had not been heated with exogenous proteases before the experiments, it was concluded that the observed cell dissociation was mediated by an endogenous serine protease. This experimental system has been used as an in vitro model of desquamation. In addition to information about the enzyme(s) involved in the cell dissociation, it has provided information about the nature of the cohesive structures in the stratum corneum. 

There is evidence that protein structures are also responsible for cell cohesion in nonpalmo-plantar stratum corneum. When punch biopsies of normal human gluteal skin were incubated in a buffer containing a mixture of the zwitterionic surfactant /V,/V,-dimethyldodecylamine and the anionic surfactant sodium dodecyl sulfate,11 there was dissociation of cells in the stratum corneum but not in the rest of the epidermis. The cell dissociation took place only in the presence of EDTA and was inhibited by the serine protease inhibitor aprotinin.12 Suzuki et al.13,14 presented evidence that spontaneous cell dissociation in nonpalmo-plantar stratum corneum could be inhibited by a combination of inhibitors of trypsin-like and chymotrypsin-like enzymes. Thus, nonpalmo-plantar stratum corneum contains endogenous proteases that mediate cell dissociation. 

There are a vast number of other factors which may be expected to influence the rate of desquamation, for instance, by affecting the rate of proteolytic reactions. pH, water, and ion concentrations, and lipid composition may all be expected to be of importance. Experimental data in this area are very scarce, but some speculations can be made. For instance, the pH dependency of SCCE activity could be of importance. SCCE has optimal activity at pH 7 to 8, but close to half its maximal activity at pH 5.5.36,37 This implies that rather small variations in either direction of the pH of the extracellular space should have effects on the rate of SCCE-mediated protein degradation. In support of this, the rate of spontaneous cell dissociation observed in plantar stratum corneum in vitro showed a marked pH dependency, being highest at neutral to weakly alkaline pH and decreasing at lower pH values.10... 

Recently, TTR1 fibrils have been decorated with the classic RGD tripeptide motif isolated from fibronectin, which encourages cell adhesion via integrin cell surface receptors (Gras et al., 2008). This bioactive tag was added to the TTR1 sequence which drives fibril assembly and the tag shown to be exposed on the fibril surface and accessible to cells following assembly. The RGD-modified fibrils were bioactive in a cell dissociation assay which measures the ability of fibrils to competitively bind to cells and induce cell detachment from a surface, as illustrated in Figure 19. In... 

It has been well established that oestrogen binds to an ER complex sedimenting at 8-10S in low-salt extracts of oestrogen responsive cells. Dissociation of this com-... 

One major challenge in hESC culture is a low survival rate after cell dissociation. Genetic manipulation (e.g., gene-targeting), and to a lesser extent, routine culture and directed differentiation are all reliant on clonal survival and/or cell dissociation. In a small screen, a selective small-molecule inhibitor of pl60-rho-associated coiled-coil kinase (ROCK), Y-27632, was found to increase hESC survival. The mechanism of action by which Y-27632 inhibits apoptosis, which is yet to be identified, should yield insights as to the causes of poor survival after dissociation (24). 

Hagiwara, S. and Kawa, K. (1984). Calcium and potassium currents in spermatogenic cells dissociated from rat seminiferous tubules. J. Physiol. 556 135-149. 

Adherent cells are released from wells using enzyme-free cell dissociation buffer (GIBCO) before capture on the magnet. Time of dissociation will vary with the cell type. Some cells are easily released after a 5 min incubation on ice in dissociation buffer while other are released more effectively at room temperature or even 37°C. 

Adherent cells can be released from the wells by incubation in enzyme-free cell dissociation buffer as detailed in Subheading 3.17. 

Benham, C.D. (1989) ATP-activated channels gate calcium entry in single smooth muscle cells dissociated from rabbit ear artery. Journal of Physiology (London), 429 689-701. 

FBS, split twice per week by incubation for 5 min with an enzyme-free cell dissociation buffer, and reseeded at a density of 1-2 x 10 cells/cm. Before an assay, the cells are seeded in methotrexate at 2 x 10 cells/well in flat-bottomed 96-well tissue culture plates, allowed to attach for 6h, washed with 250-pL/well PBS, and incubated overnight in methotrexate containing endotoxin-free BSA (0.1%, w/v). 

The spleens are disrupted with forceps or needles in lx PBS (PBS) and the cells dissociated by gentle teasing followed by filtering through a 100- and then a40-pm nylon cell strainer to remove connective tissue cells. 

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