GENEBLAZER®FRET CELL-BASED ASSAY ON CS-16 REMOVABLE CHAMBERED COVERGLASS
The GeneBLAzer® FRET Cell-based Assay provides a reliable method to study cellular pathways and cellular responses to potential drugs. The technology is based on Fluorescence Resonance Energy Transfer (FRET), a ratiometric detection method that reduces experimental artifacts enhancing assay accuracy.
The regulation and transmission of extracellular chemical signals to the nucleus are vital for DNA transcription and gene expression. The mammalian Janus Kinase signal transducers and activator of transcription (JAK/STAT) is the principal signaling mechanism for a number of cytokines and growth factors. JAK activation stimulates cell proliferation, differentiation, and apoptotic cell death. (1). Disruption of the JAK/ STAT signal transduction pathway has been shown to lead to leukemia, inflammatory disease, and erythrocytosis.
In hematopoietic cells, the JAK2/STAT5 signaling pathway plays an essential role in blood cell formation in response to cytokines such as Granulocyte-macrophage colony-stimulating factor (GM-CSF), Interleukin-3 (IL-3) and Erythropoietin (EPO)(4). In this pathway, binding of these cytokines to their respective cell surface receptors results in the activation of JAK2, which in turn phospho-activates STAT5 proteins at specific tyrosine residues(3).
TF-1 is a cell line, derived from a patient with erythroleukemia, useful for analyzing the human receptors for IL-3, GM-CSF, and EPO or the signal transduction of these hemopoietic growth factors. TF1 cells have an intact GM – CSF – JAK2 – STAT5 pathway (2). The CellSensor® irf1 – bla TF1 Cell Line contains a beta-lactamase reporter gene under control of the irf – 1 response element stably integrated into TF1 cells. CellCensor IrF1-bla TF-1 cell line was used, in this study, to assess beta-lactamase inhibition.
Intracellular Beta-Lactamase inhibition was analyzed in CellSensor® IrF1-bla TF-1 cell lines with the Live Blazer- FRET B/G substrate. A membrane permeable beta-lactamase substrate labeled with two different fluorophores was used to detect beta-lactamase activity. The two fluorophores bound to the beta-lactamase substrate, coumarin, and fluorescein, form an efficient FRET pair. In the absence of beta-lactamase activity exciting the coumarin at 409 nm in the intact molecule results in FRET (Fluorescence Resonance Energy Transfer) to the fluorescein, which emits a green fluorescence signal at 520 nm. The presence of beta-lactamase activity, however, results in cleavage of the substrate spatially separating the two dyes and disrupting FRET, so that exciting the coumarin at 409 nm now produces a blue fluorescence signal at 447 nm.
Materials and Methods
Growth media: RPMI 1640 (Gibco), 10% dialyzed FBS (Sigma-Aldrich), 1mM Sodium Pyruvate (Invitrogen), 100 U/mL Penicillin (Sigma-Aldrich), 100 μg/mL Steptomycin (Sigma-Aldrich), 5 μg/mL Blasticidin (Invitrogen), 2ng/mL Recombinant Human GM-CSF (Invitrogen), 0.1 mM non-essential aminoacids (Invitrogen).
Assay media: Opti-MEM (Gibco), 0.5% dialyzed FBS (Sigma-Aldrich), 0.1 mM Non Essential aminoacids (Invitrogen), 1mM Sodium Pyruvate (Invitrogen), 100 U/mL Penicillin (Sigma-Aldrich), 100 μg/mL Steptomycin (Sigma-Aldrich), 2 ng/mL Recombinant Human GM-CSF (Invitrogen),
LiveBLAzer-FRET B/G loading kit (Invitrogen).
Cells were cultured on a CS16 Removable Chambered CoverGlass (Grace Bio-Labs, item# 112358) at 37°C in RPMI 1640 medium supplemented with 10–20% fetal bovine serum (Invitrogen) and maintained in a humidified atmosphere of 5% CO2.
Irf-bla Cellsensor Assay
Cells were harvested from growth media, centrifuged at 400 g for 5 minutes and resuspended in assay media at a density of about 4×105 cells/mL and incubated at 37°C, 5% CO2 for 16-20 hours. Cells were then resuspended at 1 · 106 cells/ml in assay medium without blasticidin, seeded at 8 · 104 cells/well into a plated in a CS16 CultureWell Removable chambered coverglass and incubated with decreasing concentration of Potassium clavulanate, Sigma-Aldrich Corp. St. Louis, MO, USA, 0, 0.1, 10μM in 0.1% DMSO for 16 h. Following 5 to 6 h of assay incubation, 8 µL/well of 6× LiveBLAzer-FRET B/G (Invitrogen) substrate was applied to each well and incubated for 2 to 3 h at room temperature. Fluorescence was measured at 409 (ex.) nm/518 nm (em).
Visual assessment of the effects of decreasing beta-lactamase inhibitor was performed through fluorescence microscopy. The change in fluorescence due to disruption of FRET (Fluorescence Resonance Energy Transfer) is proportionate to the concentration of added inhibitor as shown in figure 1.
CS16 Removable chambered Coverglass provide the ideal surface for culturing and imaging sensitive cell lines, including stably transfected cell lines specifically engineered to study pathway specific responses. Beta-lactamase inhibition was accurately and efficiently analyzed thanks to the CS16 Removable chambered Coverglass which proved to be ideally suited for FRET- fluorescence applications since the black gasket at the bottom of the chamber helps to minimize light scatter, enhancing the fluorescence signal.
Acronyms used in the text:
JAK: Janus Kinase
STAT: Signal Transducer and Activator of Transcription
GM-CSF: Granulocyte-macrophage Colony-Stimulating Factor
DMSO: Dimethyl sulfoxide
FBS: Fetal Bovine Serum
FRET: Fluorescence Resonance Energy Transfer
(1)Jeffrey J. Babon, et al., The molecular regulation of Janus kinase (JAK) activation. Biochem J. 2014 Aug 15; 462(1): 1–13. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4112375/
(2) Kitamura T, Tange T, Terasawa T, Chiba S, Kuwaki T, Miyagawa K, Piao YF, Miyazono K, Urabe A, Takaku F. Establishment and characterization of a unique human cell line that proliferates dependently on GM-CSF, IL-3, or erythropoietin. J Cell Physiol. 1989 Aug; 140 (2):323-34. https://www.ncbi.nlm.nih.gov/pubmed?cmd=Retrieve&db=PubMed&list_uids=2663885&dopt=AbstractPlus
3) Gillinder KR, Tuckey H, Bell CC, Magor GW, Huang S, Ilsley MD, Perkins AC. Direct targets of pSTAT5 signaling in erythropoiesis. PLoS One. 2017 Jul 21; 12 (7) https://www.ncbi.nlm.nih.gov/pubmed/28732065
(4) Zhu X1,2, Liu R3,4, Guan J5, Zeng W6, Yin J2, Zhang Y2. Jak2a regulates erythroid and myeloid hematopoiesis during zebrafish embryogenesis. Int. J Med Sci. 2017 Jul 18;14 (8):758-763 https://www.ncbi.nlm.nih.gov/pubmed/28824311