HeLa cell monolayers

Structure-activity studies (116, 117) on virginiamycin Ml (90) have established the importance of the macrocyclic ring and the 13-OH group. Oxidation of the latter resulted in loss of activity, whereas the products of nonstereoselective reduction of the C-15 carbonyl group retained biological activity (116,117). Vir-giniamycins have been demonstrated to enhance lactation in ruminants (118) and to protect HeLa cell monolayers infected with Herpes simplex type I virus (119). 

Confluent HeLa cell monolayers on 6-well plates. 

After 15 h of incubation (Subheading 3.2 step 5), the HeLa cell-free mixture is treated with RNase A (20 g/ml) and RNase T1 (100 U/ml) for 30 min at room temperature, diluted to 300 pi in HESS, and added to 90% confluent monolayer of HeLa cells. Gently rock for 30 min at room temperature. In parallel, HeLa cell monolayers are infected with wt PVl (M) as control. The monolayers are washed with HESS and overlaid with 3 ml of 0.6% (w/v) gum tragacanth. After 48 h at 37°C and 5% CO, carefully remove the gum tragacanth and stain the cells with 1 ml of 1% crystal violet for 10 min. Rinse off the... 

Fig. 5. Polyacrylamide-gel electrophoresis of proteins of HeLa cells infected with UV-irradiated vaccinia virus. HeLa cell monolayers were infected with 900 particles/ cell of UV-irradiated vaccinia virus. At hourly intervals, the samples were washed with methionine-free medium and pulse labeled for 30 min with 10 xCi/ml of [ S]methionine. The radio labeled proteins were electrophoresed through a gradient (5-15%) polyacrylamide gel using the Laemmli buffer system. Lane assignments for the irradiation levels (ergs/mm ) used are lane 1, 0 lane 2, 1920 lane 3, 5760 lane 4, 9600 lane 5, 17,280 lane 6, 28,800 lane 7, cell control. From Bablanian et al. imia).

Fig. 5- Effect of salt molarity during seeding of cell monolayers with viral RNA on induction of plaque formation. Confluent HeLa cell monolayers in 60 mm petri dishes were washed with balanced salt solution, drained thoroughly and seeded with 0.1 ml of viral RNA in a Tris buffered (0.03 M, pH 8.2) solution containing the stated concentrations of KCl...

Fig. 6. Kinetics of induction of plaque formation by viral RNA in cell monolayers. HeLa cell monolayers were washed at either 36° C ( ) or 20° C (o) with Hanks balanced salt solution and seeded with viral RNA in 1 M KCl buffered with 0.03 M Tris, pH 8.2, and incubated at the stated temperatures. At certain time intervals, the monolayers were exposed to 3 jxg RNase in the RNA seeding solution (A). The interaction of the RNA with the cells was terminated by applying the agar overlay or by washing with Hanks...

To a log-phase monolayer culture of HeLa cells, add sufficient stock colchicine solution to give a final concenttation of 0.06 pg/mL. 

Figure 9.4 (a) Visual image of HeLa cells grown as a sparse monolayer on a low-e slide, (b) Raw... 

The mode of action of the harringtonines has been investigated. All inhibit protein synthesis in eukaryotic cells (190-192). The principal effect of harringtonine was inhibition of protein biosynthesis in HeLa cells (193). Homoharringtonine, a potential antineoplastic alkaloid (191), was cytotoxic in HeLa, KB, and L cells growing in monolayer cell cultures (194). 

Fig. 1. The effect of the low-molecular-weight superoxide dismutase mimic, CuDIPS, on human carcinoma (HeLa) cell growth. Monolayer cultures (0.5 x 10 6 cells/plate) were established by growth overnight in Eagles MEM medium supplemented with 10% calf serum. The medium was removed and replaced with fresh medium containing 0.1% ethanol or varying amounts of CuDIPS [copper II-(3,5-diisopropylsalicylate)2] in 0.1% ethanol. After 3 days the number of live cells per plate were assessed as described in ref. [44], The results are expressed as means of triplicate experiments s.d.

Cell-free virus or infected cells (infectious centers, ICs) can be measured at frequencies as low as 10 6. The sensitivity of the FIA rivals that of PCR, where examining the input from 106 cells is an upper limit. The FIA detects all strains of HIV, including primary isolates from patients (9,57,61,64,67-69). ICs may be detected in tissue culture cell lines and PHA blast cultures infected in vitro. The assay has also been used to quantify infectious HIV in AIDS patients (69). The assay is performed by plating either infected cells or cell-free virus on a monolayer of HeLa cells expressing CD4, CXCR4, and CCR5. Detection of clinical isolates is highly dependent on the HeLa-CD4 cell line used and is a... 

This procedure describes the preparation of nuclear and cytoplasmic extracts from cells grown in suspension (2-201) and is essentially as described by Dignam et al.1 and modified by Jamison and Garcia-Bianco.2 A procedure for small-scale preparation of extracts from HeLa cells grown as monolayer has been described by Lee and Green.3 The latter procedure is recommended when the cell material is scarce, if radioactively labelled extracts are made, if several extracts from parallel cultures, or if expensive growth conditions are necessary for cell propagation. 

Attachment of E. coli to HeLa cells. Attachment at different levels of inoculation (10 -106 CFU/mL) and physiological state of bacteria was investigated as described above. Typical results are presented in Table 1. There was no significant difference in attachment of actively growing and starved cells to the monolayer of epithelial cells. The difference in attachment between strains did not correlate with pathogenicity and was not statistically different for all investigated strains. 

To study the antiviral activity, Type 1 simplex herpes virus, KOS stock (HSV-Hg) and Indiana vesicular stomatitis virus (VSV) were used and HeLa cells were cultured in Eagle-modified Dulbecco medium (EMDM), supplemented w ith 10% foetal calf serum, on plates with 24 holes. Monolayers of these cells were infected with HSV-1 or VSV at 0.5 ufp/cell or 0.01 ufp/cell, respectively, and later the product to be assayed, pre-dissolved in DMSO, was added in concentrations of 10, 20, 50, 100 and 200 pg/ml. After 48 h incubation for HSV-1 and 24 h for VSV, at 37°C in CO2 atmosphere, the cytopathic CPE effect was measured on a phase-contrasting microscope [85],... 

Figure 9.1 Relationship between contact angle and cell adhesion. Human umbilical vein endothelial cells open circles) and HeLa cells closed circles) were allowed to attach to self-assembled monolayers (SAMs) carrying methyl (CH3), hydroxyl (OH), carboxylic acid (COOH), or amino (NH2) terminal functional groups mixed with two kinds of alkane thiols (CH3/OH, CH3/COOH and CH3/NH2) for 1 h. A clear trend showing increasing cell adhesion with decreasing contact angle was observed for aU SAMs.

A variety of cell lines, including murine-F9 embryonal-carcinoma cells, murine-melanoma cells (B16 and S9), HeLa cells and HL-60 promyelocytic leukemia cells, have been successfully used for the extraction of RARs from nuclei (1,9). For cell lines growing as monolayers, we have used three 75-cm flasks at cell densities of 5 x 10 to 10 cells per flask for a typical experiment. In the case of HL-60 cells, which grow only in suspension culture, the cells are normally maintained at densities of 0.3 x 10 to 0.9 x 10 cells/mL but may be... 

The infectivity of poliovirus is abolished by poliovirus receptor-specific monoclonal antibodies (Mab D171) and type-specific hyperimmune sera. HeLa cells are grown as monolayers in 6-well plates, washed with DMEM, and incubated with 0.5 ml of Mab D171 (final concentration 20 pg/ml) at room temperature for 2 h. Approximately 100 PFU of either sPVl (M) or wt PVl (M) is added to the cells and incubated for 1 h at room temperature. Then, the cells are washed three times with DMEM, and the number of plaques is determined after 48 h, as described above. A control plate with unrelated Mab is similarly treated. The treatment of HeLa cells with Mab 171 should completely block infection of the sPVl (M) and wt PVl (M) while the unrelated Mab had no effect on the virus infection. For the neutralization test, anti-PVl and anti-PV2 serum are incubated respectively with approximately 100 PFU of either sPVl (M) or wt PVl (M) at room temperature for 2 h. The antibody-virus mixture is added to the HeLa monolayers. Following incubation for 1 h at room temperature, the cells are washed with DMEM, and the plaques stain after 2 days, as described above. No plaque should be observed when sPVl (M) is incubated with PV type 1-specific antibodies, while serum to PV2 should not inhibit plaque formation. Altogether, these results should confirm that the de novo poliovirus virions chemically synthesized are serotype 1 and require the poliovirus receptor for infection. 

Fig. 4. Morphological changes in type C adenovirus-infected HeLa cells. (A) Uninfected HeLa cells in monolayer culture. Magnification, x 1.2 insert, x 52. (B) HeLa cell culture 3 days after Ad5 infection. Magnification, x 1.2 insert, x 52. From Periera (1958).

Culture HeLa pLuc705 cells (may be purchased from Gene Tools,USA) as exponentially growing subconfluent monolayers in DMEM (Gibco) supplemented with... 

Studies on some cell lines have shown that in tumor models such as mouse epidermal 1B6 cells and MCF-7, ROS were observed to stimulate cell growth in monolayers. In other cell lines, ROS can also be involved in the pathogenesis of cancer. By promoting cell proliferation in the transformed cancer cell lines MCF-7, HeLa, and Jurkat cells, reduced antioxidant levels were implicated in malignant transformation. Overexpression of manganese superoxide dismutase (MnSOD), a normal cellular antioxidant, enzyme was reported to revert transformation or tumor-promotion response in these and other transformed cell tines, such as human melanoma (UACC-903) cells, human breast cancer (MCF-7) cells, and mouse epidermal JB6 cells. ... 

HeLa S3 cells were maintained in monolayer culture at 37 C in Eagle s medium/10 % calf semm. Growth was synchronized by exposing the cells to hydroxyures (1 mM) for 16 h [7]. 

Stock Cell Culture. HeLa S3 cells were obtained as monolayer cultures from the Cell Culture Laboratory of the School of Public Health, University of California, Berkeley, CA. The cells were adapted to and maintained In suspension (asynchronous) by passaging them routinely In Eagle s minimum essential medium (MEM-Jokllk modified. Grand Island Biological Co. (GIBCO), Grand Island,... 

Numerous cell lines have been developed for the purpose of recombinant protein expression however, several cell lines have dominated the field. Chinese hamster ovary (CHO), NSO and SP2/0 murine myeloma, human embryonic kidney (HEK293), and COS cell fines are popular choices. HeLa, BHK, YB2/0, and PerC6 are several other available options that are not discussed in detail here. Several of these cell fines can be grown in attachment-dependent monolayers or suspension cultures. For large-scale culture, cell suspension is preferred. 

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