Rapid monoclonality verification methods to boost cell line development

<p>Regulations for cell line development are increasingly more stringent. Providing evidence that a cell line is derived from a single cell is a crucial factor in cell line generation. Typically limited dilution or fluorescence activated cell sorting is performed to seed single cells into a well. Microscopy is then used to determine the number of cells seeded in each well and monitor cell growth. While monoclonality verification via brightfield imaging is possible, debris, dust, and air bubbles make it difficult and time consuming to identify single cells which may cause high value clones to be discarded. Here we present a fluorescent method for identifying monoclonal CHO-S cells using CellTracker Green CMDFA, a fluorescent dye used to monitor cell location. We incubated CHO-S cells with Cell Tracker Green CMDFA and performed limited dilution to seed single CHO-S cells into 96-well plates. The CloneSelect Imager was used to image CHO-S cells in brightfield and fluorescence channels. Growth curve algorithms ensure that only the high growing clones are selected, while fluorescence imaging allow to quickly identify viable colonies generated from single cells. Single cell identification with the aid of fluorescence is more conclusive and robust, helping to meet stringent regulatory demands in cell line development</p

The development of automated patch clamp assays for canonical transient receptor potential channels TRPC3, 6, and 7

The canonical transient receptor potential channel subfamily (TRPC3, TRPC6, and TRPC7) contains Ca2+ permeable non-selective cation channels that are widely expressed in a variety of tissues. There is increasing evidence implicating TRPC channels, particularly TRPC3 and 6, in physiological and pathophysiological processes, eliciting interest in these channels as novel drug targets. Electrophysiology remains a benchmark technique for measuring ion channel function and accurately determining the pharmacological effects of compounds. In this report we describe the development of TRPC inhibitor assays on 2 automated planar patch clamp platforms - the IonWorks® Quattro™ and QPatch® systems. To enable activation of TRPC channels by carbachol, Chinese Hamster Ovary-K1 cells stably expressing the muscarinic M3 receptor were transduced with human TRPC3, TRPC6, or TRPC7 using BacMam viruses. TRPC3, 6, and 7 currents could be recorded on both platforms. However, the design of each platform limits which assay parameters can be recorded. Due to its continuous recording capabilities, the QPatch can capture both the activation and decay of the response. However, the transient nature of TRPC channels, the inability to reactivate and the large variation in peak currents limits the ability to develop assays for compound screening. The IonWorks Quattro, due to its discontinuous sampling, did not fully capture the peak of TRPC currents. However, due to the ability of the IonWorks Quattro to record from 64 cells per well, the variation from well to well was sufficiently reduced allowing for the development of medium-throughput screening assays. © Copyright 2014, Mary Ann Liebert, Inc. 2014

Itpkb is required for T cell development.

<p><i>Itpkb</i>^<i>+/+</i> and <i>Itpkb</i>^<i>fl/fl</i> mice were treated with tamoxifen for 5 days followed by 2 days of rest. Thymocytes and splenocytes from tamoxifen-treated mice were compared to WT and <i>Itpkb</i>^<i>-/-</i> mice via flow cytometry. (A) Gating schemes used for thymocyte analysis with antibodies to CD4, CD8, CD3, and TCRb; numbers in the plots indicate the percentages of each gated population. (B) Total numbers of each thymocyte subset. (C) Gating scheme for analysis of the splenocytes stained with antibodies to CD4, CD8, CD3, and B220; numbers in the plots indicate the percentages of each gated population. (D) Total numbers of the indicated splenocyte populations. DP: double positive. Data from one representative experiment is shown. *, P < 0.05; **, P < 0.01.</p

Ins(1,3,4,5)P4 inhibits Orai1-mediated current.

<p>HEK293-Orai1-Stim1 cells in serum-free media were placed on the QPatch HT recording system. Following cell break-in (1), little current was measured. Orai1 currents were then activated in a time-dependent manner following passive store depletion with an EGTA/BAPTA solution to chelate intracellular Ca^<i>2+</i> (2), and then stabilized in an open state (3). Next, (4), Ins(1,3,4,5)P4 (A), a control Ins(1,4,5,6)P4 (B), or 0.1% DMSO (C) was applied for 15 minutes. At the end of each experiment, 2-APB (5) was applied to block any remaining Orai1 current. In (C), percent inhibition values are expressed as the difference in current amplitude before compound application and following 2-APB application (% inhibition = ((base pA–compound pA) / (base pA– 2-APB pA)) X 100. The numbers in parentheses indicate the number of cells tested for each condition on 2 separate experimental days. Data shown is representative of three independent experiments. *, P < 0.05; **, P < 0.01</p

Itpkb is required for T cell function by negatively regulating SOC channels.

<p>WT and <i>Itpkb</i>^<i>fl/fl</i> mice were immunized with either the T-independent antigen, TNP-Ficoll, or the T-dependent antigen DNP-KLH. ELISA of TNP-specific IgG3 (A) or DNP-specific IgG1 (B) antibody responses on day 12 following immunization. Data from one representative experiment is shown (**, P < 0.01). <i>In vitro</i>, Itpkb-deficient mature lymphocytes are diminished in their proliferative capacity as measured by thymidine incorporation of purified CD4^<i>+</i> cells following stimulation with various concentrations of anti-CD3 plus anti-CD28 (C). The data from one representative experiment are shown with values representing the mean counts per minute (cpm) ± SEM. *P < 0.05. Analysis of Ca^<i>2+</i> responses using the Ca^<i>2+</i> sensitive dyes Fluo-4 and Fura Red were evaluated after cross-linking the antigen receptor either in the presence or absence of exogenous Ca^<i>2+</i>. Splenocytes gated on CD4 were treated with anti-CD3-biotin, followed by cross-linking with streptavidin (S.A.) in the presence of exogenous calcium (top panel), or in the absence of exogenous calcium, followed by calcium re-addition to examine SOC channel function (bottom panel). Ionomycin (Iono.) stimulation was used at the end of each run to control for equivalent dye loading. Data is shown as the mean fluorescent ratio of Fluo-3 and Fura-Red. Representative data of three independent experiments are shown.</p

Itpkb inhibitors block rat antigen-induced arthritis.

<p>(A) Lewis rats were immunized intra-dermally on Day -21 and -14 with methylated BSA (mBSA), followed by daily oral dosing of GNF362, or dexamethasone (Dex) as a positive control. On Day 0, the rats received an intra-articular injection of mBSA into the right knee joint. (B) Serum was sampled on Day +7, and IgG antibody titers to mBSA were determined by ELISA. The antibody titers were calculated by determining the average dilution at which half-maximal absorbance is detected after subtraction of background. Fold reduction in antibody titer over vehicle is shown in the table. Data shown is one representative experiment. (C) The diameters of the right and left knees were measured on Days 0, 2, 4, and 7, and the ratio of right over left knee diameters (R/L) is shown. (D) Histological analysis of the knee joint was performed blindly and scored on a 5-point scale at the termination of the study. Data shown is one representative experiment. *, P < 0.05; **, P < 0.01.</p

Itpkb negatively regulates pro-apoptotic gene expression and AICD in CD4^<i>+</i> T cells.

<p>Either purified (primary) CD4^<i>+</i> T cells (A) or in vitro-expanded (secondary) CD4^<i>+</i> T cells (B) were stimulated with anti-CD3/28 beads for the indicated time points. Following RNA isolation, cDNA was generated, and subjected to real-time quantitative PCR analysis for Bim, Bcl2, Fas, and FasL. The Y-axis represents the respective transcript normalized to either B2-microglobulin or GAPDH values determined for each sample. One representative experiment is shown with the mean values of each genotype ± SE. *, P < 0.05; **, P < 0.01. (C) Purified CD4^<i>+</i> cells were labeled with CFSE and stimulated with anti-CD3/28 beads in the presence of anti-FasL or an isotype control Ig. 72 hours following stimulation, cells were stained with Annexin V and DAPI. Numbers in the lower left quadrant indicate the percentage of live cells at the time of analysis. (D) Proliferation measured by CFSE dilution was followed after 72 hours in culture. Blue and red lines indicate stimulated and unstimulated cells, respectively. Numbers above bracketed lines indicate the percentage of divided cells. Data shown is representative of three independent experiments.</p

Identification and characterization of Itpkb inhibitors.

<p>(A) GNF362 was identified following a 2 million compound biochemical screen, co-crystallography with Itkpb, and additional medicinal chemistry optimization. (B) The biochemical activity of GNF362 was determined in Kinase Glo assays using purified Itpka, Itpkb, and Itpkc proteins [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131071#pone.0131071.ref014" target="_blank">14</a>]. Data shown is one representative experiment. (C) The cellular activity of GNF362 was profiled on wild type B cells loaded with the calcium-sensitive dye, Fluo-4. Splenocytes were pre-incubated with varying concentrations of GNF362, and SOC-mediated Ca^<i>2+</i> entry, depicted as the mean value of Fluo-4 over time, was measured on the FLIPR following stimulation with anti-IgM and calcium add-back. (D) The peak calcium response, after calcium re-addition is shown as a function of the concentration of GNF362 in the assay, with an EC50 of 12nM. Data shown is one representative experiment. (E) Purified CD4^<i>+</i> T cells were stimulated in the presence of GNF362, along with the addition of either anti-FasL or a control Ig, to determine the effect on proliferation and FasL-mediated activation-induced cell death. Data shown is representative of three independent experiments. (F) Wild type mice were dosed orally with GNF362 at 3, 10, or 25mg/kg twice daily for 9 days. The percentage of CD4^<i>+</i> T cells in the thymus was determined by FACS analysis. Data shown is representative of three independent experiments. **, P < 0.01.</p

Overexpression of TRPC5 can also confer sensitivity to englerin A.

<p>(<b>A</b>) Expression of TRPC4 and TRPC5 in the cell lines from the CLiP experiment. Each circle represents a single cell line and expression levels were measured by microarray (TRPC4 probe 220818_s_at, TRPC5 probe 220552_at). The circle representing the DMS-79 cell line is indicated. (<b>B</b>) The effect of englerin A on cell viability in HEK293T cells transiently transfected with a vector expressing TRPC5 (mean +/- standard deviation).</p

TRPC4 expression is necessary and sufficient for cell proliferation effects of englerin A.

<p><b>(A</b>) Effect of TRPC4 siRNA knockdown on viability of A-498 cells in the presence of englerin A. An siRNA targeting luciferase was used as a control (mean+/- S.E.M.) Percent reduction of the TRPC4 mRNA levels are indicated in the legend, KD stands for knockdown. TRPC4 mRNA levels were normalized to peptidyl prolyl isomerase A (PPIA) mRNA levels. (<b>B</b>) Effect of TRPC4 siRNA knockdown on viability of A-673 cells in the presence of englerin A (mean +/- S.E.M.) (<b>C</b>) Effect of overexpression of TRPC4 by transient transfection on viability of HEK293T cells in the presence of englerin A. TRPC4 expression vector concentrations are indicated by different shapes (<b>D</b>) Effect of TRPC4 expression on cell viability in the presence of an englerin A in HEK293T cells engineered to express TRPC4 under control of a Doxycycline (Dox) regulated promoter (mean +/- standard deviation). 100 ng/ml Dox (black circles), 0 ng/ml Dox (open circles). (<b>E</b>) Western blot visualizing the levels of TRPC4 in the presence or absence of 100 ng/ml Dox. (<b>F</b>) Effect of PKCtheta inhibitor compound 27 on response to englerin A in A-498 cells. (<b>G</b>) Effect of PKCtheta inhibitor compound 27 on response to englerin A in A-673 cells.</p