Gluc as a reporter in mammalian cells.

<p>(A) Schematic representation of the expression cassettes for Gluc-IRES-CFP cloned in the CSCW lentivirus vector. (B) High infection rate of cells with lentivirus vectors (M.O.I. = 30) as monitored by cerulean fluorescence. Scale bar, 100 µm. (C) Levels of Gluc activity in cells vs. medium vs. cells+medium. 293T cells were infected with the lentivirus vector carrying the expression cassette for Gluc-IRES-CFP and 20,000 cells were plated in wells of a 96-well plate. 48 hrs post-infection, new medium was added to the wells and Gluc activity was measured 24 hrs later in conditioned medium, viable washed cells or cells+conditioned medium after adding 2.5 µM coelenterazine.</p

Linearity and sensitivity of the Gluc assay.

<p>(A) 293T cells were infected with the lentivirus vector carrying the expression cassette for Gluc-IRES-CFP and 20,000 cells were plated in wells of 96-well plate. Gluc activity was monitored over time in 10 µL of conditioned medium after addition of 20 µM coelenterazine. The release of Gluc to the conditioned medium is linear with time. (B) 293T cells were co-transfected with pHGC-Fluc and either pHGC-Gluc or pSEAP expression plasmids. Gluc and SEAP activities were assayed in conditioned medium 24 hrs later. The signal over background ratio (S/B) values, normalized to the intracellular levels of Fluc, are plotted against the number of cells. The Gluc assay can detect a single cell with S/B of 40 whereas the SEAP assay requires 20,000 cells to get similar S/B under similar assay conditions. The mean ± S.E.M. is presented on the graphs (n = 3).</p

Sensitive Assay for Mycoplasma Detection in Mammalian Cell Culture

Mycoplasma contamination in mammalian cell cultures is often overlooked yet is a serious issue which can induce a myriad of cellular changes leading to false interpretation of experimental results. Here, we present a simple and sensitive assay to monitor mycoplasma contamination (mycosensor) based on degradation of the <i>Gaussia</i> luciferase reporter in the conditioned medium of cells. This assay proved to be more sensitive as compared to a commercially available bioluminescent assay in detecting mycoplasma contamination in seven different cell lines. The <i>Gaussia</i> luciferase mycosensor assay provides an easy tool to monitor mammalian cell contaminants in a high-throughput fashion

Monitoring of ER stress with Gluc.

<p>293T cells expressing Gluc were subjected to no or different concentrations of DTT (A–D) or thapsigargin (E–F) to induce ER stress. (A) Real-time RT-PCR for spliced XBP-1 mRNA which increased in response to >1 mM DTT 4 hrs after treatment. Fold induction is calculated with respect to the control non-infected/non-treated cells. (B&F) Western blot analysis showing upregulation of BiP and/or phosphorylated eIF2alpha levels in response to ER stress 24 hrs after treatment. Blot is also probed with Gluc antibody as well as beta-tubulin antibody for equal loading. (C&E) Conditioned medium assayed for Gluc activity 4 hrs after DTT or thapsigargin treatment showing that Gluc secretion is decreased in response to ER stress. (D) Untreated or treated cells with 1 mM DTT were monitored overtime for the level of Gluc secretion by assaying an aliquot of the conditioned medium for Gluc bioluminescence. The mean ± S.E.M. is presented on the graphs (n = 3), with *p≤0.01 as calculated by the student's t-Test.</p

Gluc-YFP fusion to visualize secretory pathway in real-time.

<p>(A) Schematic representation of the Gluc-YFP fusion cloned in the lentivirus vector. (B) Uninfected 293T cells or cells infected with a lentivirus vector expressing Gluc-YFP fusion were lysed and analyzed by western blotting with anti-Gluc antibody. (C) Cells expressing Gluc-YFP fusion were treated with BFA or nocodazole and their conditioned medium were assayed for Gluc activity 24 hrs later. *p≤0.01 as predicted by student T-test. (D) Fluorescence microscopy of a single live cell expressing Gluc-YFP and either untreated or treated with BFA showing that this fusion is trapped in the ER upon BFA treatment. Scale bar, 10 µm.</p

Gluc as a reporter to monitor secretory pathway.

<p>293T cells were infected with the lentivirus vector expressing Gluc and were plated in wells of 96-well plate. Cells were treated for 24 hrs with different drugs which interfere with the secretory pathway. (A) Cell-free conditioned media were assayed for Gluc activity which showed that Gluc secretion is decreased upon blocking the secretory pathway. *p≤0.01 as predicted by student T-test. (B) Immunocytochemistry on cells expressing Gluc with and without treatment with BFA showing that Gluc (cy3, red) co-localizes with the ER marker PDI (Alexa488, green). Scale bar, 10 µm.</p

Multiplex Blood Reporters for Simultaneous Monitoring of Cellular Processes

Reporters secreted into the conditioned medium of cells in culture or into blood in vivo have shown to be useful tools for simple and noninvasive monitoring of biological processes in real-time. Here, we characterize the naturally secreted <i>Vargula</i> luciferase as a secreted blood reporter and show that this reporter can be multiplexed with the secreted <i>Gaussia</i> luciferase and alkaline phosphatase for simultaneous monitoring of three different cellular processes in the same biological system. We applied this system to monitor the response of three different subsets of glioma cells to a clinically relevant chemotherapeutic agent in the same well in culture or animal in vivo. This system could be extended to any field to detect multiple processes in the same biological system and is amenable for high-throughput screening to find drugs that affect multiple cellular populations/phenomena simultaneously

Enhanced delivery of IL-6 by MSC transplants compared to MSC conditioned medium.

<p>(A) Serum profiles of human IL-6 after IV administration of concentrated conditioned medium into C57Bl/6 mice. The plot was normalized to the dose of conditioned medium that was contributed by 1×10⁶ cells. Pharmacokinetic parameters (B) Cmax, (C) AUC, (D) Tmax, (E) Half-life, and (F) Elimination constant were calculated for IL-6 exposure by cell transplants compared to CM administration. Significant differences between cell transplants compared to CM whereby higher levels of IL-6 and longer artificial duration was observed in plasma after cell transplantation. Time points for serum analyses were 0.5, 8, and 24 hours after cell or media injection. Mice were serially analyzed as batches of N = 5 per group. * denotes P>0.01.</p

Combined pharmacokinetic monitoring of MSCs and MSC-secreted IL-6.

<p>(A) Bioluminescent images of C57Bl/6 mice over a period of three days after IV cell administration of one million luciferase-engineered human MSCs. (B) Photon flux of bioluminescent signal over time after IV cell administration. Durable BLI signals were detected up to 24 hours in mice that were injected IV with MSCs. (C) Serum ELISA measurements of human IL-6 released by IV cell transplants over time. Time points for serum and imaging analyses were 0.5, 8, 24, and 72 hours after cell injection. Pooled mouse serum was serially analyzed as batches of N = 5.</p

Golgi-dependent secretion mechanism of MSC-derived IL-6 in vivo.

<p>Brefeldin A pre-treatment of MSCs was used to evaluate blockade of IL-6 release in vitro and in vivo. (A) MTT assay of MSCs treated at different concentrations of brefeldin. A non-toxic dose of 5 ug/ml was used for functional studies. (B) Human IL-6 levels in vitro after brefeldin pre-treatment. Significant reduction in 24 hour release of IL-6 was observed across all doses. (C) Alteration in serum IL-6 delivery by MSCs pretreated with a Golgi-apparatus inhibitor, Brefeldin A. MSCs were incubated with 5 µg/ml of BFA for one day and then injected into C57Bl/6 mice and compared to untreated MSCs in terms of serum IL-6 delivery. Brefeldin treatment of MSCs led to diminished release of human IL-6 in vitro and in vivo. (D) Area-under-curve analysis of human IL-6 after MSC pre-treatment with brefeldin A and transplantation. Exposure to IL-6 was significantly reduced by inhibition of the Golgi apparatus. Time points for serum analyses were 0.5, 8, and 24 hours after cell injection. Mice were serially analyzed as batches of N = 5 per group. * denotes P>0.01.</p