Improved Methods for Preserving Macromolecular Structures and Visualizing Them by Fluorescence and Scanning Electron Microscopy

To determine the optimal procedures to preserve cytoskeletal and other macromolecular structures for microscopic studies we have evaluated the effects of various methods to extract cultured cells. In this report, we compare results using different fixatives, crosslinking reagents, and permeabilization methods on (1) the labeling of cells for fluorescence microscopy with phalloidin or antibody against tubulin; and (2) the morphological preservation of macromolecular structures for scanning electron microscopy. Maximal labeling of F-actin with phalloidin was obtained by fixing cells in 4% para formaldehyde (PFA) and labeling the unextracted cells with methanolic phalloidin, whereas maximal labeling of tubulin required prefixation with either PFA or the bi functional protein crosslinking reagent, dithiobis (succinimidylpropionate) (DSP) and extraction with ethanol or Triton in a high salt buffer. However, for both qualitative and quantitative light and electron microscopic studies of intracellular macromolecular structures, prefixation with DSP and extracting with Triton X-100 in a stabilizing buffer is the overall method of choice for both labeling and morphological studies. Although other methods provide maximal labeling or preservation of specific structures, this method provides excellent preservation of morphological structure while allowing proteins to be preserved and labeled by specific probes

Actin filament reorganization in HL-60 leukemia cell line after treatment with G-CSF and GM-CSF.

Currently, information regarding the influence of growth factors on the cytoskeleton, including G-CSF and GMCSF, remains limited. In the present study we show alterations in F-actin distribution and cell cycle progression in HL-60 promyelocytic leukemia cells, resulting from treatment with these cytokines in vitro. We found that both agents caused F-actin reorganization. Although multiple potential effects of various growth factors have been described previously, in our experimental conditions, we observed some rather subtle differences between the effects of G-CSF and GM-CSF on studied cells. The presence of these cytokines in the cell environment caused not only increased F-actin labeling in the cytoplasm, but also a weaker intensity of peripheral ring staining in comparison with control cells. In spite of the fact that HL60 cells exposed to G-CSF and GM-CSF contained different F-actin structures such as aggregates and F-actin network, the rate of actin polymerization was not significantly enhanced. Moreover, alterations were mainly related to considerable changes in the relative proportion of these different structures, what might be reflected by specific features of the differentiation process, with regard to the kind of stimulating factor used. Thus, reorganization of F-actin and other results obtained in our experimental conditions, might reflect unique characteristics of the differentiation process in HL-60 cells, involving low apoptosis frequency, the G1 to S phase transition in the cell cycle, as well as possible alternative ways of the cell death

Effect of Bacterial Products on Oral Wound Healing and Gingival Fibroblasts

Jenny Ji ; Psychology; jenny.ji‐[email protected]. Faculty mentor: Barbara Safiejko‐Mroczka; Biology; [email protected]

Delayed Closure of Oral Wounds in the Presence of Prophylactic Antibiotics

Julia Murray ; Biology; julia.c.murray‐[email protected]. Faculty mentor: Barbara Safiejko‐Mroczka; Biology; [email protected]

Actin filament reorganization in HL-60 leukemia cell line after treatment with G-CSF and GM-CSF.

Currently, information regarding the influence of growth factors on the cytoskeleton, including G-CSF and GMCSF, remains limited. In the present study we show alterations in F-actin distribution and cell cycle progression in HL-60 promyelocytic leukemia cells, resulting from treatment with these cytokines in vitro. We found that both agents caused F-actin reorganization. Although multiple potential effects of various growth factors have been described previously, in our experimental conditions, we observed some rather subtle differences between the effects of G-CSF and GM-CSF on studied cells. The presence of these cytokines in the cell environment caused not only increased F-actin labeling in the cytoplasm, but also a weaker intensity of peripheral ring staining in comparison with control cells. In spite of the fact that HL60 cells exposed to G-CSF and GM-CSF contained different F-actin structures such as aggregates and F-actin network, the rate of actin polymerization was not significantly enhanced. Moreover, alterations were mainly related to considerable changes in the relative proportion of these different structures, what might be reflected by specific features of the differentiation process, with regard to the kind of stimulating factor used. Thus, reorganization of F-actin and other results obtained in our experimental conditions, might reflect unique characteristics of the differentiation process in HL-60 cells, involving low apoptosis frequency, the G1 to S phase transition in the cell cycle, as well as possible alternative ways of the cell death