HeLa-S3 Cell Growth Conditions in Serum-Free Medium and Adaptability for Proliferation in Suspension Culture

Abdeen, Shenf H.; Abdeen, Ahmed; El‐Enshasy, Hesham A.; Shereef, Abdalla A. El

doi:10.3923/jbs.2011.124.134

Cited by 7 publications

(6 citation statements)

References 16 publications

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“…The HeLa S3 cell line (CCL-2.2, ATCC) was adapted for suspension growth. 28 Cells were cultured in DMEM/F12 (1:1 ratio) medium with 4.5 g/L glucose, 2 mM glutamine supplemented with 10% fetal bovine serum. The cell cultures were maintained at 37 °C in a humidified 5% CO 2 atmosphere and with gentle shaking at 200 rpm to keep the cells suspended.…”

Section: Cell Culturementioning

confidence: 99%

A High-Throughput Dose-Response Cellular Thermal Shift Assay for Rapid Screening of Drug Target Engagement in Living Cells, Exemplified Using SMYD3 and IDO1

McNulty

Bonnette

et al. 2018

SLAS Discovery

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A persistent problem in early small-molecule drug discovery is the frequent lack of rank-order correlation between biochemical potencies derived from initial screens using purified proteins and the diminished potency and efficacy observed in subsequent disease-relevant cellular phenotypic assays. The introduction of the cellular thermal shift assay (CETSA) has bridged this gap by enabling assessment of drug target engagement directly in live cells based on ligand-induced changes in protein thermal stability. Initial success in applying CETSA across multiple drug target classes motivated our investigation into replacing the low-throughput, manually intensive Western blot readout with a quantitative, automated higher-throughput assay that would provide sufficient capacity to use CETSA as a primary hit qualification strategy. We introduce a high-throughput dose-response cellular thermal shift assay (HTDR-CETSA), a single-pot homogenous assay adapted for high-density microtiter plate format. The assay features titratable BacMam expression of full-length target proteins fused to the DiscoverX 42 amino acid ePL tag in HeLa suspension cells, facilitating enzyme fragment complementation-based chemiluminescent quantification of ligand-stabilized soluble protein. This simplified format can accommodate determination of full-dose CETSA curves for hundreds of individual compounds/analyst/day in replicates. HTDR-CETSA data generated for substrate site and alternate binding mode inhibitors of the histone-lysine N-methyltransferase SMYD3 in HeLa suspension cells demonstrate excellent correlation with rank-order potencies observed in cellular mechanistic assays and direct translation to target engagement of endogenous Smyd3 in cancer-relevant cell lines. We envision this workflow to be generically applicable to HTDR-CETSA screening spanning a wide variety of soluble intracellular protein target classes.

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Section: Cell Culturementioning

confidence: 99%

A High-Throughput Dose-Response Cellular Thermal Shift Assay for Rapid Screening of Drug Target Engagement in Living Cells, Exemplified Using SMYD3 and IDO1

McNulty

Bonnette

et al. 2018

SLAS Discovery

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“…Approximate cell diameter values could be determined from the microscopy images of cells inside the capillary (Figure S2). HeLa cells have been reported to have average diameters of ∼17 μm; however, we observed smaller diameters (average ∼12 μm) and a more spherical shape for HeLa cells cultured in suspension in our experiments . Images were taken immediately following successful cell loading to ensure an accurate representation of cell size since the cells were observed to swell shortly after entering the capillary due to the start of lysis in the 75% FA loaded into the capillary.…”

Section: Resultsmentioning

confidence: 85%

On-capillary Cell Lysis Enables Top-down Proteomic Analysis of Single Mammalian Cells by CE-MS/MS

et al. 2022

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Proteomic analysis of limited samples and single cells requires specialized methods that prioritize high sensitivity and minimize sample loss. Consequently, sample preparation is one of the most important steps in limited sample analysis workflows to prevent sample loss. In this work, we have eliminated sample handling and transfer steps by processing intact cells directly in the separation capillary, online with capillary electrophoresis coupled to tandem mass spectrometry (CE-MS/MS) for top-down proteomic (TDP) analysis of low numbers of mammalian cancer cells (<10) and single cells. We assessed spray voltage injection of intact cells from a droplet of cell suspension (∼1000 cells) and demonstrated 0−9 intact cells injected with a dependency on the duration of spray voltage application. Spray voltage applied for 2 min injected an average of 7 ± 2 cells and resulted in 33−57 protein and 40−88 proteoform identifications (N = 4). To analyze single cells, manual cell loading by hydrodynamic pressure was used. Replicates of single HeLa cells (N = 4) lysed on the capillary and analyzed by CE-MS/MS demonstrated a range of 17−40 proteins and 23−50 proteoforms identified. An additional cell line, THP-1, was analyzed at the single-cell level, and proteoform abundances were compared to show the capabilities of single-cell TDP (SC-TDP) for assessing cellular heterogeneity. This study demonstrates the initial application of TDP in single-cell proteome-level profiling. These results represent the highest reported identifications from TDP analysis of a single HeLa cell and prove the tremendous potential for CE-MS/MS on-capillary sample processing for high sensitivity analysis of single cells and limited samples.

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“…was found to be around 20% v/v for HeLa cell lines. HeLa cells and other human cancer-derived cell lines are commonly employed in medical studies for assessing the efficacy of chemotherapies, anticancer agents, drug quality control and testing the expression of recombinant proteins [47,48]. Kaba and Egorova [49] have indicated the significant toxicity of AgNPs toward the HeLa and U937 tumor cells.…”

Section: Discussionmentioning

confidence: 99%

Fusarium mangiferae as New Cell Factories for Producing Silver Nanoparticles

Hamzah¹,

Salah²,

Maroof³

2018

Journal of Microbiology and Biotechnology

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Finding a safe and broad-spectrum medication is a goal of scientists, pharmacists, and physicians, but developing and fabricating the right medicine can be challenging. The current study describes the formation of silver nanoparticles (AgNPs) by Fusarium mangiferae. It involves the antibiofilm activity of the nanoparticles against Staphylococcus aureus. It also involves cytotoxic effect against mammalian cell lines. Well-dispersed nanoparticles are formed by F. mangiferae. The sizes of the nanoparticles were found to range from 25 to 52 nm, and UV-Vis scan showed absorption around 416-420 nm. SEM, TEM, and AFM results displayed spherical and oval shapes. Furthermore, the FTIR histogram detected amide I and amide II compounds responsible for the stability of AgNPs in an aqueous solution. AgNPs were observed to decrease the formation of biofilm at 75% (v/v). DNA reducing, smearing, and perhaps fragmentation were noticed after treating the bacterial cells with 50% (v/v). Additionally, cell lysis was detected releasing proteins in the supernatant. It was also observed that the AgNPs have the ability to cause 59% cervical cancer cell line (HeLa) deaths at 25% (v/v), however, they showed about 31% toxicity against rat embryo fibroblast transformed cell lines (REF). The results of this study prove the efficiency of AgNPs as an antibiofilm against S. aureus, suggesting that AgNPs could be an alternative to antibiotics. It must also be emphasized that AgNPs displayed cytotoxic behavior against mammalian cell lines. Further studies are needed for assessing risk in relation to the possible benefit of prescribing AgNPs.

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HeLa-S3 Cell Growth Conditions in Serum-Free Medium and Adaptability for Proliferation in Suspension Culture

Cited by 7 publications

References 16 publications

A High-Throughput Dose-Response Cellular Thermal Shift Assay for Rapid Screening of Drug Target Engagement in Living Cells, Exemplified Using SMYD3 and IDO1

A High-Throughput Dose-Response Cellular Thermal Shift Assay for Rapid Screening of Drug Target Engagement in Living Cells, Exemplified Using SMYD3 and IDO1

On-capillary Cell Lysis Enables Top-down Proteomic Analysis of Single Mammalian Cells by CE-MS/MS

Fusarium mangiferae as New Cell Factories for Producing Silver Nanoparticles

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