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Archive for the ‘Celigo User Publications’ Category

Celigo Helping Move Research Forward to Find a Cure for Alzheimer’s

Dmitry Kuksin | November 7, 2017 | No Comments | Celigo User Publications

November is National Alzheimer’s Disease Awareness Month and research into a cure for the disease is vigorous. In a recently published paper “CRISPR/Cas9-Correctable mutation-related molecular andphysiological phenotypes in iPSC-derived Alzheimer’s PSEN2N141I neurons” researchers used the Celigo image cytometer upstream of CRISPR/Cas9 to visualize and asses cell death related to the sensitivity of iPSC-derived PSEN2N1411 neurons in the presence of Aβ42 oligomer toxicity.

Celigo assists in optimizing CHO cells for biopharmaceutical production

Sara Barker | February 11, 2016 | No Comments | Celigo User Publications

The Novo Nordisk Center for Biosustainability (Denmark) set out to improve the efficiency of Chinese hamster ovary (CHO)-cell based production of non-monoclonal antibody, therapeutic glycoproteins designed to serve as biopharmaceuticals. To optimize the growth and production capacities of these CHO cells, the scientists looked at: lipid-based transfection, cell cultivation, cell counting, and antibody-independent product titer. Different growth and transfection parameters were investigated to see which yielded the highest growth profiles and production capacities. The Celigo was used in combination with Hoechst and propidium iodide to count the cells in 96-well format. The system developed here miniaturized the process and allowed for efficient, higher throughput evaluation of target genes aimed at enhancing therapeutic glycoprotein production by CHO cells.

Read the full publication here. 

Celigo evaluates quinomycin A as a possible therapeutic tool for pancreatic cancer

Sara Barker | January 26, 2016 | No Comments | Celigo User Publications

The Notch-disrupting and cancer stem cell-inhibiting effects of the drug quinomycin A were investigated at the University of Kansas Medical Center. Using human pancreatic cancer cells PanC-1, MiaPaCa-2, and BxPC-3 and the Celigo to determine the number and size of pancreatospheres, researchers evaluated the drug’s ability to block cancer stem cell growth via inhibition of the Notch signaling pathway. After administration of the drug, proliferation and colony formation were blocked in cancer cell lines but not in normal pancreatic epithelial cells. Furthermore, cancer stem cell markers were reduced as was pancreatosphere formation. This work identifies quinomycin A as an efficacious inhibitor of pancreatic cancer stem cells.

Read the full publication here. 

The Celigo image cytometer can be used in a wide array of applications and experiments. Visit our website to learn more about how the Celigo could benefit your research.

Celigo furthers studies of homologous recombination DNA repair machinery

Sara Barker | January 21, 2016 | No Comments | Celigo User Publications

The Danish Cancer Society Research Center recently published a study furthering their analysis of homologous recombination DNA repair machinery. The group previously reported on a growth factor, PSIP1, that enables DNA end resection. With GFP-transfected U2OS cells, the group investigated a structurally similar protein, hepatoma-derived growth factor-related protein 2 (HDGFRP2). The Celigo analyzed cell number and viability via fluorescent markers. The group reports that HDGRFP2 may help to repair silent genes that have been impaired or active genes inhibited by DNA damage.

Read the full publication here. 

Celigo evaluates a plant extract for glioblastomoa multiforme treatment

Sara Barker | December 1, 2015 | No Comments | Celigo User Publications

At the Canary Center at Stanford for Early Cancer Detection, investigators studied how AshwaMAX (a steroidal lactone from a winter cherry plant, Withania somnifera, extract) might work as an oral treatment for those with the highly aggressive cancer glioblastoma multiforme (GBM). A heterogeneous disease, non-specific therapies for GBM have proven largely ineffective. Two patient-derived GBM lines (GBM2, GBM39) and one GBM cell line were cultured to create neurospheres that were then exposed to various concentrations of AshwaMAX.  Celigo measured cell proliferation and cell death via Trypan Blue staining. AshwaMAX inhibited the neurospheres at nanomolar concentrations. After additional work in vivo, researchers concluded that AshwaMAX is a viable candidate as a GBM therapy.

Read the full publication here. 

The Celigo image cytometer is a versatile instrument with a best in class bright field and 3 channels of fluorescence.  To learn about the different ways Celigo could be used in your research, please visit our website or contact one of our technical scientists at info@nexcelom.com

Ignyta tests Celigo against Cell Titer-Glo for cell proliferation

Sara Barker | October 23, 2015 | No Comments | Celigo User Publications

Here’s a great example of how the Celigo image cytometer is able to perform common experiments while saving time and money!

Ignyta, Inc. was looking for a new way to perform reagent-free proliferation analyses with suspension cells. This new method had to produce results which correlated well to their current method, Cell Titer-Glo®. Nexcelom and Ignyta partnered to perform a head-to-head cell proliferation comparison between Celigo® and Cell Titer-Glo. Using four suspension cell types (Ba/F3 parental cell line, Ba/F3 expressing an oncogenic gene, oncogenic gene mutant A and B), Ignyta plated all cells at a concentration of 5,000 cells/well in the presence of various concentrations of four drugs (1-4). Three days later, Celigo evaluated cell proliferation using bright field to capture and analyze whole-well images in less than 5 minutes per plate. The exact same plates were then used to perform the standard (“lyse and read”) Cell Titer-Glo assay.

The two methods produced IC50 values that were highly comparable across all cell types and drug concentrations (r2=0.998). Celigo proved to be a rapid, accurate analysis for Ignyta’s suspension cells, providing images for visual verification and a reagent-free method which meant the same cells could be analyzed repeatedly over a time course. Now, Ignyta had a simplified process by which to analyze cell proliferation, one that was highly correlated to their previous method and required no reagents or additional incubation times, saving time, cost, and supplies.

Read the full publication here!

If you are interested to discuss how the Celigo could benefit your research, contact us at info@nexcelom or visit our website for more information.

Nexcelom’s Celigo Detects CRISPR Transfection Efficiency of sgRNA in CHO Cells

Leo Chan | March 30, 2015 | No Comments | Celigo User Publications

Introduction

Chinese hamster ovary (CHO) cells are the cell type of choice for biopharmaceuticals due to their propensity to correctly fold and post-translationally modify human proteins [1]. Here, researchers use an optimized bioinformatics tool (“CRISPy”) to generate chimeric RNA called “single guide” RNA (sgRNA) in order to disrupt FUT8, a gene that is utilized in N-glycosylation. This novel web-based tool ensures efficient, fast, and low-cost genetic manipulation of CHO cells for future biopharmaceutical applications.

Materials and Methods

Cell culture and transfection of CHO cells

CHO cells were cultured and transfected by Nucleofector Kit V with 1 μg of Cas9 plasmid and 1 μg of sgRNA plasmid. Two days later, cells were transfected with pmaxGFP and assessed for transfection efficiency using Nexcelom’s Celigo.

Phenotypic analysis of FUT8 knockout cells

Five days post-transfection, the selection agent Lens culinaris agglutinin (LCA) was added. (LCA binds plasma membrane proteins, which leads to downstream cell death. LCA serves as a selection agent for FUT8 knockout cells as LCA cannot bind to cells devoid of FUT8.) Cells were stained for phenotypic analysis with Hoechst (red in image) and fluorescein-labeled LCA (F-LCA; green in image). FUT8 wild-type cells will stain green, while FUT8 knockout cells will not. Bright-field and fluorescent images were captured with the Celigo.

*Note:  For more detailed Materials and Methods and a complete account of the entire study, please refer to the original manuscript [http://onlinelibrary.wiley.com/doi/10.1002/bit.25233/abstract]

Results

  • LCA selection caused control cells to become non-adherent and rounded.
  • Cells transfected with Cas9 and sgRNA remained adherent, indicating the successful knockout of FUT8 in those cells.
  • Cells transfected with Cas9 and sgRNA without LCA selection showed that F-LCA negative cells represented 29.1% of the entire population.
  • Cells transfected with Cas9 and sgRNA with LCA selection showed 98.6% of cells were F-LCA negative, as demonstrated by other studies proving the lack of functional FUT8 [2-4].
Figure 1A-C. Phenotypic analysis of FUT8 knockout cells. Cells were transfected with either Cas9 plasmid alone or Cas9 plasmid plus sgRNA. At Day 6 the bright field images were taken (A). On Day 13 the cells were stained with Hoechst (red) and fluorescein-labeled LCA (green). Quantification of the images for F-LCA positive (wild-type FUT8) and F-LCA negative (FUT8 knockout) can be seen in C.

Figure 1A-C. Phenotypic analysis of FUT8 knockout cells. Cells were transfected with either Cas9 plasmid alone or Cas9 plasmid plus sgRNA. At Day 6 the bright field images were taken (A). On Day 13 the cells were stained with Hoechst (red) and fluorescein-labeled LCA (green). Quantification of the images for F-LCA positive (wild-type FUT8) and F-LCA negative (FUT8 knockout) can be seen in C.

Conclusions

  • This RNA-guided CRISPR Cas9 application successfully disrupted genes in CHO cells.
  • The web-based bioinformatics design tool “CRISPy” provides an effective, low-cost method for genetically manipulating CHO cells.

References

  1. Jayapal, K.P., et al., Recombinant protein therapeutics from CHO cells – 20 years and counting. Chem Eng Prog, 2007. 103: p. 40-47.
  2. Malphettes, L., et al., Highly efficient deletion of FUT8 in CHO cell lines using zinc-finger nucleases yields cells that produce completely nonfucosylated antibodies. Biotechnol Bioeng, 2010. 106(5): p. 774-83.
  3. Mori, K., et al., Engineering Chinese hamster ovary cells to maximize effector function of produced antibodies using FUT8 siRNA. Biotechnol Bioeng, 2004. 88(7): p. 901-8.
  4. Yamane-Ohnuki, N., et al., Establishment of FUT8 knockout Chinese hamster ovary cells: an ideal host cell line for producing completely defucosylated antibodies with enhanced antibody-dependent cellular cytotoxicity. Biotechnol Bioeng, 2004. 87(5): p. 614-22.

 

Investigation of IAPP Role in Increasing ROS Production and Apoptosis in p53-deficient Tumor Cells using Celigo Imaging Cytometer

Leo Chan | December 23, 2014 | No Comments | Celigo User Publications

The entire family of tumor protein p53 (TP53) enhances functions such as apoptosis and autophagy in normal cellular functioning. TP53 is a tumor repressor gene that is often inactivated in human cancers. Reactivating p53 has proven difficult to achieve therapeutically, however. Researchers at MD Anderson Cancer Center are investigating other members of the p53 pathway in order to elucidate new therapeutic options to suppress p53-deficient tumor growth.

ΔN isoforms of two members of the p53 family, p63 and p73, are usually overexpressed in cancers and these isoforms (which lack the acidic transactivation domain) act on p53 in a dominant-negative fashion, blocking its tumor repression abilities [1-6]. Using transfected human cancer cell lines, researchers were able to investigate the roles ΔNp63 and ΔNp73 play in promoting tumor survival. Deleting these ΔN isoforms of p63 and p73 produced tumor regression via the upregulation of IAPP, an amylin-encoding gene that is secreted with insulin. A synthetic analogue of amylin, pramlintide, has proven successful in blocking tumor survival, so this IAPP pathway was analyzed as a new option for treating p53-deficient cancers [7]. Researchers uncovered that TAp63 and TAp73 (isoforms with acidic transactivation domains) transcriptionally regulate IAPP, which in turn blocks glycolysis in the tumor cells, thereby decreasing tumor cell survival [8].

Using Nexcelom’s Celigo, researchers investigated the downstream effects of IAPP, which demonstrated increased rates of apoptosis and the production of reactive oxygen species (ROS) through inhibition of glycolysis [9, 10].

Materials and Methods

*for information regarding the transfection of H1299 cells with ΔNp63, ΔNp73, or IAPP, or the applications of siRNA, please see Venkatanarayan, et al. (2014) “IAPP-driven metabolic reprogramming induces regression of p53 tumours in vivo.” Nature. doi:10.1038/nature13910; http://www.nature.com/nature/journal/vaop/ncurrent/nature13910/metrics

Pramlintide Treatment

Twelve hours after plating, cells were treated with 10μg/mL pramlintide acetate (AMYLIN Pharmaceuticals) or placebo for 48 hours.

Apoptosis Assay

Cells were plated in 96-well plates. After 12 hours, cells were washed with annexin-binding buffer and a mixture of annexin V-Alexa Fluor 488 propidium iodide (PI) and Hoechst 33342. All images were analyzed for percent apoptosis by the Celigo.

ROS Assay

Cells were plated in 96-well plates. After 12 hours, the cells were incubated with CellROX Deep Red Reagent and Hoechst 33342 for 45 minutes. All images were analyzed for percent ROS by the Celigo.

NAC Treatment

Twelve hours after plating, cells were treated with NAC 2mM final concentration for a period of 1 hour.

Results

  • In human lung adenocarcinoma cells (H1299), the rates of ROS and apoptosis, the latter measured by annexin V/PI staining, increased in cells expressing IAPP or after treatment with pramlintide. In comparison, p53-deficient cells containing ΔNp63 or ΔNp73 showed no reduction in ROS or apoptosis.
IAPP induces ROS and apoptosis in H1299 cells

Figure 1a-f. IAPP induces ROS and apoptosis in H1299 cells. Both the knock down of ΔNp63 or ΔNp73 (through siRNA), the expression of IAPP, or treatment with pramlintide increased ROS (c and d) and induced apoptosis (a-top, e, and f). (The addition of N-acetyl-L-cysteine [NAC+, bottom or NAC-, top] blocks ROS and demonstrated that inhibiting ROS in those cells also blocked apoptosis.)

  • When media from H1299 cells transfected with ΔNp63 or ΔNp73 and containing secreted IAPP was added to naïve H1299, the cells demonstrated higher levels of apoptosis, as measured by annexin V/PI staining, as well as increased ROS.
Secreted IAPP from ΔNp63 or ΔNp73 transfected H1299 cells

Figure 2a and b. Secreted IAPP from ΔNp63 or ΔNp73 transfected H1299 cells was capable of inhibiting glycolysis in naïve H1299 cells, thereby increasing the percentage of ROS (a) and apoptosis (b) positive cells.

Conclusion

  • Exploiting the IAPP pathway may yield novel methods of treating p53-deficient cancers.

*Note:  the paper outlining these experiments in more detail may be found here [http://www.nature.com/nature/journal/vaop/ncurrent/nature13910/metrics]

References

  1. Flores, E.R., et al., Tumor predisposition in mice mutant for p63 and p73: evidence for broader tumor suppressor functions for the p53 family. Cancer Cell, 2005. 7(4): p. 363-73.
  2. Su, X., et al., TAp63 suppresses metastasis through coordinate regulation of Dicer and miRNAs. Nature, 2010. 467(7318): p. 986-90.
  3. Su, X., D. Chakravarti, and E.R. Flores, p63 steps into the limelight: crucial roles in the suppression of tumorigenesis and metastasis. Nat Rev Cancer, 2013. 13(2): p. 136-43.
  4. Tomasini, R., et al., TAp73 knockout shows genomic instability with infertility and tumor suppressor functions. Genes Dev, 2008. 22(19): p. 2677-91.
  5. Yang, A., et al., p63, a p53 homolog at 3q27-29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities. Mol Cell, 1998. 2(3): p. 305-16.
  6. Su, X., et al., TAp63 is a master transcriptional regulator of lipid and glucose metabolism. Cell Metab, 2012. 16(4): p. 511-25.
  7. Edelman, S., H. Maier, and K. Wilhelm, Pramlintide in the treatment of diabetes mellitus. BioDrugs, 2008. 22(6): p. 375-86.
  8. Castle, A.L., et al., Amylin-mediated inhibition of insulin-stimulated glucose transport in skeletal muscle. Am J Physiol, 1998. 275(3 Pt 1): p. E531-6.
  9. Mattson, M.P. and Y. Goodman, Different amyloidogenic peptides share a similar mechanism of neurotoxicity involving reactive oxygen species and calcium. Brain Res, 1995. 676(1): p. 219-24.
  10. Schubert, D., et al., Amyloid peptides are toxic via a common oxidative mechanism. Proc Natl Acad Sci U S A, 1995. 92(6): p. 1989-93.