Ratiometric [Ca2+]i Measurements in Adherent Cell-Lines Using the NOVOstar Microplate Reader

2012 ◽  
pp. 111-120 ◽  
Author(s):  
Benjamin D. Hunt ◽  
David G. Lambert
Keyword(s):  
Cell Lines ◽  
Adherent Cell ◽  
Microplate Reader

Isolation of single cells from adherent cell lines using Smart Aliquotor CE v1 (protocols.io.bhjmj4k6)

protocols.io ◽  
2020 ◽  
Author(s):  
Lucy Kimbley ◽  
Rachel Parker ◽  
Maaike Sybil ◽  
John Holloway ◽  
Emily Swindle ◽  
...  
Keyword(s):  
Cell Lines ◽  
Single Cells ◽  
Adherent Cell

scholarly journals Megakaryocytic cell line-specific hyperploidy by cytotoxic necrotizing factor bacterial toxins

Blood ◽  
1996 ◽  
Vol 88 (9) ◽  
pp. 3465-3473 ◽  
Author(s):  
KM Hudson ◽  
NC Denko ◽  
E Schwab ◽  
E Oswald ◽  
A Weiss ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Phorbol Ester ◽  
Dna Content ◽  
Nuclear Dna ◽  
Nuclear Dna Content ◽  
Adherent Cell ◽  
Target Cells ◽  
Suspension Cells ◽  
Cytotoxic Necrotizing Factor

Cytotoxic necrotizing factor (CNF) toxins, isolated from certain Escherichia coli strains known to cause intestinal and extra intestinal infections, induce reorganization of the actin cytoskeleton and generate hyperploidy in adherent cell lines. We have examined the effect of CNF toxin on one of the few cell types that naturally increase nuclear DNA content, megakaryocytes. Our studies show that only hematopoietic cells capable of differentiating along the megakaryocyte lineage responded to the CNF2 toxin by becoming polyploid and by reorganizing actin. The K562, HEL, and CHRF-288–11 cell lines can be induced with phorbol ester to differentiate along the megakaryocyte lineage, and these cells also respond to the toxin with increased DNA content and actin cytoskeletal rearrangements. Interestingly, treatment of the K562 and HEL cell lines with CNF2 does not result in an increase in production of the megakaryocytic marker glycoprotein IIIa, unlike phorbol ester treatment. Conversely, two T-cell leukemic cell lines, CEM and Molt4, and the promyelocytic HL-60 cell line, which do not differentiate along the megakaryocyte lineage in response to phorbol myristate acetate, do not respond to CNF2, by increased expression of gpIIIa, increased nuclear DNA content, or actin reorganization. A potential target of these toxins, RhoA, is expressed by both megakaryocytic and nonmegakaryocytic cell lines, as shown by reverse transcription-polymerase chain reaction and Western blot. Although it is clear that the CNF toxins can affect a wide variety of adherent nonhematopoietic cell lines, we propose that the response to CNF, in terms of reorganizing actin structure and increase in DNA content in hematologic suspension cells, correlates with the capability of these target cells to differentiate along the megakaryocytic lineage.

Enhancement of stress-induced apoptosis in B-lineage cells by caspase-9 inhibitor

Blood ◽  
2004 ◽  
Vol 104 (9) ◽  
pp. 2873-2878 ◽  
Author(s):  
Nisha Shah ◽  
Rebecca J. Asch ◽  
Alana S. Lysholm ◽  
Tucker W. LeBien
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Leukemic Cell ◽  
Adherent Cell ◽  
Cell Contact ◽  
Caspase 9 ◽  
B Lineage ◽  
B Lineage Cells

Abstract We have established human B-lineage (BLIN) acute lymphoblastic leukemia cell lines that retain a dependency on fibroblast monolayers for survival and proliferation. Eight hours following removal from adherent cell contact BLIN cells undergo a decrease in mitochondrial transmembrane potential and an increase in annexin V binding. Unexpectedly, the caspase-9 inhibitor (C9i) benzyloxycarbonyl-Leu-Glu-His-Asp-fluoromethylketone enhanced the appearance of apoptotic cells within 8 hours following removal of BLIN cells from fibroblast monolayers. C9i enhancement of apoptosis was dose dependent and did not occur with irreversible inhibitors of caspases-2, -3, -6, and -8. C9i also enhanced apoptosis in cord blood-derived CD19+ B-lineage cells (but not myeloid cells) removed from murine stromal cells. Longer exposure (> 18 hours) to C9i culminated in apoptosis in a panel of B-lineage acute lymphoblastic leukemia (ALL) cell lines in the presence or absence of fibroblast monolayers, as well as in 2 proliferating leukemic cell lines (RAMOS and CEM). BLIN-4L cells made deficient in caspase-9 by RNA interference exhibited no resistance to apoptotic signals and actually showed increased apoptotic sensitivity to staurosporine. These collective results suggest that a 4-amino acid caspase inhibitor of caspase-9 can promote apoptosis and that at least some types of apoptotic pathways in B-lineage ALL do not require caspase-9.

Procedures to test for the serum-free cultivation of adherent cell lines on microcarriers

1983 ◽  
Vol 8 (4) ◽  
pp. 161-165 ◽  
Author(s):  
Berthold G. D. Bödeker ◽  
Guy J. Berg ◽  
Guy Hewlett ◽  
H. Dieter Schlumberger
Keyword(s):  
Cell Lines ◽  
Adherent Cell ◽  
Serum Free

Characterisation of four merkel cell carcinoma adherent cell lines

1995 ◽  
Vol 60 (1) ◽  
pp. 100-107 ◽  
Author(s):  
J. Helen Leonard ◽  
Peter Dash ◽  
Peter Holland ◽  
John H. Kearsley ◽  
John R. Bell
Keyword(s):  
Cell Carcinoma ◽  
Cell Lines ◽  
Merkel Cell Carcinoma ◽  
Adherent Cell ◽  
Merkel Cell

scholarly journals Oxidized Juncuenin B Analogues with Increased Antiproliferative Activity on Human Adherent Cell Lines: Semisynthesis and Biological Evaluation

2020 ◽  
Vol 83 (11) ◽  
pp. 3250-3261
Author(s):  
Csaba Bús ◽  
Ágnes Kulmány ◽  
Norbert Kúsz ◽  
Tímea Gonda ◽  
István Zupkó ◽  
...  
Keyword(s):  
Cell Lines ◽  
Antiproliferative Activity ◽  
Biological Evaluation ◽  
Adherent Cell

scholarly journals Structures of the free and inhibitors-bound forms of bromelain and ananain from Ananas comosus stem and in vitro study of their cytotoxicity

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Mohamed Azarkan ◽  
Erik Maquoi ◽  
François Delbrassine ◽  
Raphael Herman ◽  
Nasiha M’Rabet ◽  
...  
Keyword(s):  
Cell Lines ◽  
Complex Mixture ◽  
In Vitro Study ◽  
Cysteine Proteases ◽  
Ananas Comosus ◽  
Adherent Cell ◽  
High Discrimination ◽  
Protease Substrate ◽  
Case Sequence

Abstract The Ananascomosus stem extract is a complex mixture containing various cysteine ​​proteases of the C1A subfamily, such as bromelain and ananain. This mixture used for centuries in Chinese medicine, has several potential therapeutic applications as anti-cancer, anti-inflammatory and ecchymosis degradation agent. In the present work we determined the structures of bromelain and ananain, both in their free forms and in complex with the inhibitors E64 and TLCK. These structures combined with protease-substrate complexes modeling clearly identified the Glu68 as responsible for the high discrimination of bromelain in favor of substrates with positively charged residues at P2, and unveil the reasons for its weak inhibition by cystatins and E64. Our results with purified and fully active bromelain, ananain and papain show a strong reduction of cell proliferation with MDA-MB231 and A2058 cancer cell lines at a concentration of about 1 μM, control experiments clearly emphasizing the need for proteolytic activity. In contrast, while bromelain and ananain had a strong effect on the proliferation of the OCI-LY19 and HL-60 non-adherent cell lines, papain, the archetypal member of the C1A subfamily, had none. This indicates that, in this case, sequence/structure identity beyond the active site of bromelain and ananain is more important than substrate specificity.

Microtubule depolymerization inhibits clathrin coated-pit internalization in non-adherent cell lines while interleukin 2 endocytosis is not affected

1997 ◽  
Vol 110 (19) ◽  
pp. 2441-2447 ◽  
Author(s):  
A. Subtil ◽  
A. Dautry-Varsat
Keyword(s):  
Cell Lines ◽  
Interleukin 2 ◽  
Lymphoid Cells ◽  
Membrane Receptors ◽  
Adherent Cell ◽  
Transferrin Receptors ◽  
Hemopoietic Cell ◽  
Microtubule Depolymerization ◽  
Microtubule Cytoskeleton ◽  
Microtubule Disruption

The microtubule cytoskeleton is generally not considered to be essential for the first steps of clathrin-mediated endocytosis of membrane receptors. Its role in clathrin-independent endocytosis has not been investigated. We have previously shown that the cytokine interleukin 2 (IL2) is internalized in lymphoid cells expressing its receptors when clathrin-dependent endocytosis is inhibited. Here we compare the internalization of IL2 and of transferrin, a marker of clathrin-dependent endocytosis, after microtubule disruption. In hemopoietic cell lines, which express IL2 receptors, transferrin receptor entry was inhibited by about 40%. However, in adherent cell lines, transferrin entry was unaffected by microtubule disruption, as previously reported. Unlike the case for transferrin, internalization of IL2 receptors was not affected by depolymerization of the microtubule cytoskeleton in hemopoietic cell lines. These results show that IL2 and transferrin receptors do not have the same endocytic properties and support our previous conclusion that these receptors follow different pathways of endocytosis.

CYTOTOXICITY AND CELLULAR UPTAKE OF CELLULOSE NANOCRYSTALS

Nano LIFE ◽  
2012 ◽  
Vol 02 (03) ◽  
pp. 1241006 ◽  
Author(s):  
SHUPING DONG ◽  
ANJALI A. HIRANI ◽  
KATELYN R. COLACINO ◽  
YONG WOO LEE ◽  
MAREN ROMAN
Keyword(s):  
Drug Delivery ◽  
Cell Lines ◽  
Cellular Uptake ◽  
Exposure Time ◽  
Cellulose Nanocrystals ◽  
Drug Carriers ◽  
Raw 264.7 ◽  
Cytotoxic Effects ◽  
Microplate Reader ◽  
Ldh Assay

There is growing evidence that filamentous nanoparticles offer advantages over spherical ones in drug delivery applications. The purpose of this study was to assess the potential of rod-like, plant-derived cellulose nanocrystals (CNCs) for nanomedical uses. Besides a nonspherical morphology, their facile bioconjugation, surface hydrophilicity and small size render CNCs promising drug carriers. The cytotoxicity of CNCs against nine different cell lines (HBMEC, bEnd.3, RAW 264.7, MCF-10A, MDA-MB-231, MDA-MB-468, KB, PC-3 and C6) was determined by MTT and LDH assay. CNCs showed no cytotoxic effects against any of these cell lines in the concentration range and exposure time studied (0–50 μg/mL and 48 h, respectively). Cellular uptake of fluorescein-5′-isothiocyanate-labeled CNCs by these cell lines, quantified with a fluorescence microplate reader, was minimal. The lack of cytotoxicity and the low nonspecific cellular uptake support our hypothesis that CNCs are good candidates for nanomedical applications.

scholarly journals Wortmannin Potentiates Integrase-Mediated Killing of Lymphocytes and Reduces the Efficiency of Stable Transduction by Retroviruses

2001 ◽  
Vol 21 (4) ◽  
pp. 1164-1172 ◽  
Author(s):  
René Daniel ◽  
Richard A. Katz ◽  
George Merkel ◽  
James C. Hittle ◽  
Tim J. Yen ◽  
...  
Keyword(s):  
Dna Repair ◽  
Protein Kinase ◽  
Cell Lines ◽  
Reporter Genes ◽  
Adherent Cell ◽  
Protein Kinase Activity ◽  
Human Immune Deficiency Virus ◽  
Nhej Pathway ◽  
Dna Repair Proteins ◽  
Cellular Dna

ABSTRACT Retroviral infection induces integrase-dependent apoptosis in DNA-PK-deficient murine scid lymphocytes. Furthermore, the efficiency of stable transduction of reporter genes is reduced in adherent cell lines that are deficient in cellular DNA-repair proteins known to mediate nonhomologous end joining (NHEJ), such as DNA-PK and XRCC4 (R. Daniel, R. A. Katz, and A. M. Skalka, Science 284:644–647, 1999). Here we report that wortmannin, an irreversible inhibitor of phosphatidylinositol 3-kinase (PI-3K)-related PKs, including the catalytic subunit of DNA-dependent protein kinase (DNA-PKCS) and ATM, sensitizes normal murine lymphocytes to retrovirus-mediated cell killing. We also show that the efficiency of stable transduction of reporter genes in human (HeLa) cells, mediated by either an avian sarcoma virus or a human immune deficiency virus type 1 vector, is reduced in the presence of wortmannin. The dose dependence of such reduction correlates with that for inhibition of PI-3K-related protein kinase activity in these cells. Results from wortmannin treatment of a panel of cell lines confirms that formation and/or survival of transductants is dependent on components of the NHEJ pathway. However, stable transduction is virtually abolished by wortmannin treatment of cells that lack ATM. These results suggest that ATM activity is required for the residual transduction observed in the NHEJ-deficient cells. Our studies support the hypothesis that DNA repair proteins of the NHEJ pathway and, in their absence, ATM are required to avoid integrase-mediated 2killing and allow stable retroviral DNA transduction. The studies also suggest that cells can be sensitized to such killing and stable retroviral DNA integration blocked by drugs that inhibit cellular DNA repair pathways.

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