Kyle Gee

Fast, cell-compatible click chemistry with copper-chelating azides for biomolecular labeling.

We report that azides capable of copper-chelation undergo much faster “Click chemistry” (copper-accelerated azide-alkyne cycloaddition, or CuAAC) than nonchelating azides under a variety of biocompatible conditions. This kinetic enhancement allowed us to perform site-specific protein labeling on the surface of living cells with only 10–40 µM CuI/II and much higher signal than could be obtained using the best previously-reported live-cell compatible CuAAC labeling conditions. Detection sensitivity was also increased for CuAAC detection of alkyne-modified proteins and RNA labeled by metabolic feeding.

Low-Affinity Ca2+ Indicators Compared in Measurements of Skeletal Muscle Ca2+ Transients

The low-affinity fluorescent Ca(2+) indicators OGB-5N, Fluo-5N, fura-5N, Rhod-5N, and Mag-fluo-4 were evaluated for their ability to accurately track the kinetics of the spatially averaged free Ca(2+) transient (Delta[Ca(2+)]) in skeletal muscle. Frog single fibers were injected with one of the above indicators and, usually, furaptra (previously shown to rapidly track Delta[Ca(2+)]). In response to an action potential, the full duration at half-maximum of the indicators fluorescence change (DeltaF) was found to be larger with OGB-5N, Fluo-5N, fura-5N, and Rhod-5N than with furaptra; thus, these indicators do not track Delta[Ca(2+)] with kinetic fidelity. In contrast, the DeltaF time course of Mag-fluo-4 was identical to furaptras; thus, Mag-fluo-4 also yields reliable kinetic information about Delta[Ca(2+)]. Mag-fluo-4s DeltaF has a larger signal/noise ratio than furaptras (for similar indicator concentrations), and should thus be more useful for tracking Delta[Ca(2+)] in small cell volumes. However, because the resting fluorescence of Mag-fluo-4 probably arises largely from indicator that is bound with Mg(2+), the amplitude of the Mag-fluo-4 signal, and its calibration in Delta[Ca(2+)] units, is likely to be more sensitive to variations in [Mg(2+)] than furaptras.

Novel live alkaline phosphatase substrate for identification of pluripotent stem cells.

Alkaline phosphatases (AP) are a class of enzymes that hydrolyze phosphate containing molecules under alkaline conditions. In humans, there are primarily four different types of this enzyme; intestinal, placental, placental-like and non-tissue specific forms. The non-tissue specific isozyme of AP is expressed in liver, bone and kidney. A similar isozyme was identified in pluripotent stem cells when monoclonal antibodies, TRA-2-49/6E, recognizing determinants of human embryonal carcinoma (EC) cells showed specific reactivity to this isoform.1 AP is also known to be expressed at high levels in other pluripotent stem cell types such as embryonic germ cells (EG), embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC).2–5 Although definitive measures of pluripotency involves in-vitro tri-lineage differentiation and in-vivo teratoma formation, the most widely tested and validated panel for initial evaluation of ESC and iPSC consists of Stage Specific Embryonic Antigen SSEA4; Tumor Rejection Antigens TRA-1-60, TRA-1-81; AP; Oct4 and Nanog.6, 7 n nIn the case of murine ESC, AP positive colony forming in-vitro assay is used as a measure of pluripotency to demonstrate the ability of cells to single cell clone, attach and proliferate.8 A similar assay has been adapted for hESC where the sensitivity of the AP positive Colony forming assay to detect loss of pluripotent hESC has been found to be more sensitive than marker expression.9 More recently, the onset of AP positive colonies during early stages of reprogramming is used as an initial indicator of successful reprogramming of cells. Furthermore, in some instances the number of AP positive colonies is used as a mark of reprogramming efficiency.10 Nevertheless, this marker alone is not a definitive marker for the established iPSC clones. Additional marker evaluation is necessary to identify and qualify bona fide iPSCs.11 AP expression levels is a less sensitive measure to differentiate between undifferentiated and early differentiating cells since its expression level is reported to be varied depending on the lineage of differentiation.12 AP staining has been used as a fast and easy method that results in a specific chromogenic or fluorescent staining of the pluripotent stem cells. However, the current methods using AP staining require cell fixation and/or result in end products that accumulate within the cells. As a result, these AP stained colonies often lose their morphology and cannot be propagated any further. Inability to further culture selected pluripotent colonies identified using AP staining is a serious disadvantage of this methodology. An ideal solution would be an AP substrate that stains cells without altering the integrity or characteristics of stem cells thereby allowing further expansion of the stained colonies. n nHere in, we report the development and application of a novel fluorogenic live cell permeant substrate for AP (Live AP Stain). When incubated with cells for 20–30xa0min in basal culture media, this stain shows specific and robust staining of pluripotent cells such as human EC, murine and human ESC and iPSC with minimal or no staining of feeder cells and human fibroblasts. Stained colonies retain their morphology and preserve their cell health. The green fluorescence of the stained colonies is eliminated from cells 60–90xa0min after removal of the stain from the media. We have further utilized this stain in iPSC work flow to identify emerging iPSC clones during reprogramming of BJ human fibroblasts using CytoTune™; a Sendai-virus based non-integrating reprogramming method.13 Clones with robust AP staining were manually picked and propagated further. Expanded clones expressed other pluripotent markers, differentiated into cell types representative of the three germ layers and maintained a normal karyotype. These results indicate that AP Live Stain reported in this study does not alter the integrity or characteristics of the stained cells and is therefore an ideal tool to label early intermediates during iPSC generation or clonal populations of ESC for further selection and expansion.

Fluorogenic cephalosporin substrates for β-lactamase TEM-1.

Cephalosporin was used to synthesize soluble and precipitating fluorogenic β-lactam substrates that demonstrated differential catalytic hydrolysis by three different subtypes of β-lactamase: TEM-1 (class A), p99 (class C), and a Bacillus cereus enzyme sold by Genzyme (class B). The most successful soluble substrate contained difluorofluorescein (Oregon Green 488) ligated to two cephalosporin moieties that, therefore, required two turnovers to produce the fluorescent Oregon Green 488 leaving group. The bis-cephalosporin modification was required so that the final reaction product was the Oregon Green 488 carboxylic acid rather than a less bright phenolic adduct of the dye. Hydrolysis in pH 5.5 Mes and pH 7.2 phosphate-buffered saline (PBS) buffers was similar, but in pH 8.0 Tris the hydrolysis rate nearly doubled. Activity of the β-lactamases on the various substrates was shown to depend highly on the linker between the cephalosporin and the fluorophore, with an allyl linker promoting faster turnover than a phenol ether linker. Measured K(m) values for dichlorofluorescein and difluorofluorescein cephalosporin substrates were approximately the same as K(m) values for penicillin G and ampicillin found in the literature (~30-40μM).

Development and in vitro characterization of ratiometric and intensity-based fluorescent ion sensors.

Fluorescent ion sensors are quite valuable in experimental biology. The development of new sensor molecules requires determination of spectral properties (absorption bands, fluorescence excitation, and emission maxima) in order to characterize the type of optical response to the target ion. This optical response type and magnitude are used, in combination with solutions of buffered ion of precisely manipulated concentration, to determine the in vitro affinity for the target ion. Buffered aqueous ion solutions of appropriate pH and ionic strength are necessary to predict the performance of new sensors in biological applications. A series of novel benzoxazole-BAPTA calcium sensors, in addition to Rhod-3 (a new version of rhod-2), are described and their optical responses to calcium ion characterized.

Abstract 5098: GFP compatibility with EdU cell proliferation assay

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CAnnExamining cell proliferation in GFP expressing cells is of general interest in many aspects of biology including regenerative medicine, stem cells, developmental biology and some fields of cancer research. However, visualization of GFP expression is not readily compatible with commonly used proliferation assays which incorporate a thymidine analog to directly measure S-phase proliferation of DNA. With the BrdU (bromo-deoxyuridine) assay, an antibody based detection method, alcohol for fixation and hydrochloric acid for DNA denaturation is commonly used. Neither chemical is compatible with GFP fluorescence. For imaging applications there are some methods to avoid the use of HCl as a denaturant with the BrdU assay, however; they are typically “home-brew” methods used to partially digest the DNA, adding extra steps, and not easily performed.nnThe much faster and reliable EdU (ethynyl-deoxyuridine) cell proliferation assay which uses click chemistry for detection of S-phase proliferation of DNA, uses formaldehyde based fixation and avoids the use of HCl for DNA denaturation. However, the use of copper to catalyse the click reaction also negatively affects GFP fluorescence. Anti-GFP antibodies can be used in the click reaction work flow, to “retrieve” the lost GFP fluorescence but are not convenient and add extra steps.nnWe present recent improvements to the click chemistry based EdU cell proliferation assay which minimizes the loss of GFP and other fluorescent proteins signals and avoids the need for “work around” methods. The resulting click reaction is both more rapid and brighter than the “classic” click EdU assay. The modifications preserve most of the GFP fluorescence and permit multiplex detection with EdU with no change in the work flow of the classic click EdU assay.nnWe optimized components in the improved click reaction conditions and tested compatibility with various fluorescent proteins. Examples of click chemistry and GFP/RFP/mCherry compatibility are presented using the EdU cell proliferation assay in both cell culture and in GFP expressing tissue. Additionally, improvements are demonstrated in with other applications of click chemistry assays used for imaging as well as with on flow cytometry platforms where GFP, R-phycoerythrin (R-PE), or other fluorescent proteins are commonly combined with cell proliferation assays.nnThe use of the modified click reaction is an enabling improvement over originally described copper based click reactions and will further enhance the utility of EdU based cell proliferation.nnCitation Format: Scott T. Clarke, Zhichao Song, Kelvin Y. Kwan, Carolyn DeMarco, Aleksey Rukavishnikov, Upinder Singh, Kyle Gee. GFP compatibility with EdU cell proliferation assay. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5098. doi:10.1158/1538-7445.AM2014-5098