Calcium assay to simultaneously monitor vehicle- and drug-treated cells.

<p>Expanded human T cells were treated with vehicle (ethanol) or Gαi inhibitor, pertussis toxin (PTX) for 1 hour at 37°C. Vehicle and PTX treated cells were stained for surface antigens, loaded with 1 μM Fura Red, AM and stained with a cell viability dye. Chemokine stimulated calcium flux was monitored simultaneous in the vehicle and PTX groups by selective gating. (A) Schematic of staining protocol and data acquisition by flow cytometry. (B) Representative example of selective gating strategy to monitor live cells, singlets, CCR6⁺, distinguishing vehicle- and PTX-treated cells based on anti-CD3 antibodies conjugated to distinct fluorochromes (AlexaFluor700 or APC-Cy7). Numbers next to gated population indicate percentage of cells expressing antigen of interest. (C) Ratiometric analysis of Fura Red calcium dye depicted as mean Fura Red Ratio over time. Plots depict calcium flux by CCR6⁺ T cells in response to CCL20 (125 ng/ml), or ionomycin (5 μg/ml). Stimulus was added at 25 seconds. (D) Data from C analyzed as the proportion of cells producing a calcium signal greater than the average background signal. One representative experiment of n = 3 shown, performed with a minimum of two technical replicates.</p

Analysis of calcium flux by discrete populations of primary cells.

<p>Expanded human T cells were stained for surface CCR6 protein, loaded with 1 μM Fura Red, AM and stained with a cell viability dye. Calcium flux in response to CCL20 (125 ng/ml) or ionomycin (5 μg/ml) was monitored by flow cytometry. (A) Gating strategy to exclude non-viable cells, select singlets, and distinguish CCR6⁺ from CCR6^- cells. (B) Calcium flux depicted as the mean value of the Fura Red Ratio over time, in response to CCL20 or ionomycin. (C) Calcium response depicted as the percent of cells responding to stimuli greater than the average background signal. One representative experiment of n = 5 shown, performed with a minimum of two technical replicates.</p

Ratiometric analysis of Fura Red calcium dye and Fura Red titration.

<p>Expanded human T cells were loaded with increasing concentrations of Fura Red, AM for 30 minutes. Intracellular calcium mobilization in response to ionomycin (5 μg/ml) was measured by flow cytometry. Arrows indicate the addition of ionomycin. (A) Gating parameters to select lymphocytes (based upon forward and side scatter properties) and singlets. (B) Intracellular calcium mobilization detected as a rise in fluorescence measured off the Violet laser (406 nm) and decreasing fluorescence measured off the Green laser (532nm). (C) Ratiometric analysis of Fura Red signal and dye titration. Fura Red Ratio calculated as emission off the Violet laser over emission off the Green laser. Values depict the mean of the ratio over time. One representative experiment of n = 2 shown.</p

Identifying channels available in combination with Fura Red calcium dye.

<p>Expanded human T cells were loaded with 1 μM Fura Red, AM for 30 minutes. Calcium flux in response to ionomycin (5 μg/ml added at 25 seconds) was recorded on all available channels using an LSRII SORP flow cytometer. Cells were selectively gated on lymphocytes and singlets. The names assigned to the different detectors represent fluorochromes that are commonly detected in that channel. (A) Channels used to perform ratiometric analysis of Fura Red dye. (B) Representative examples of channels that display a shift in MFI following calcium mobilization. Channels such as these cannot be used for surface protein characterization in combination with Fura Red. (C) Channels with no change in MFI following calcium mobilization. Such channels are available for surface marker and cell viability analysis in combination with Fura Red dye. On the tested LSRII SORP, four available channels were identified, and fluorochromes commonly detected in these channels include: AlexFluor488, APC, APC-Cy7, and AlexaFluor700. One representative experiment of n = 2 shown.</p

Calcium flux monitored in whole PBMC.

<p>Fresh PBMC were stained for surface markers CD3, CD14, CD16 and CCR2 and loaded with 1 μM Fura Red, AM. (A) Forward and side scatter properties were used to distinguish myeloid and lymphoid cells. (B) Monocyte subsets were distinguished on the basis of CD14 and CD16 expression. (C) Histogram depicts CCR2 expression by monocyte populations. (D) Real time calcium flux in response to CCL2 (65 ng/ml) added at 25 seconds, monitoring distinct monocyte populations. (E) T lymphocytes were identified by CD3 expression. (F) Histogram depicts CCR2 expression by T lymphocytes. (G) Real time calcium flux in response to CCL2 (65 ng/ml), monitoring CD3⁺ T lymphocyte subsets. The number next to selective gates indicates the proportion of cells among the parent population. One experiment of n = 4 shown, performed with three technical replicates.</p

Schematic overview of study design.

<p>Following the start of the study period, 1^st January 2010, 4 theoretical qualifying events are shown. Within the 30 day follow up period, a re-attendance in hospital is observed for 1) an episode with oral treatment and 2) an episode with IV treatment. Each initial attendance enters into either the oral or IV group and a subsequent re-attendance may or may not occur during the 30 day follow-up period.</p

Baseline characteristics for patients at initial attendance.

<p>Baseline characteristics for patients at initial attendance.</p

The pathogen recognition sensor, NOD2, is variably expressed in patients with pulmonary tuberculosis-4

<p><b>Copyright information:</b></p><p>Taken from "The pathogen recognition sensor, NOD2, is variably expressed in patients with pulmonary tuberculosis"</p><p>http://www.biomedcentral.com/1471-2334/7/96</p><p>BMC Infectious Diseases 2007;7():96-96.</p><p>Published online 16 Aug 2007</p><p>PMCID:PMC2018706.</p><p></p>erial strains [A = combined data and B to F individual data]. NOD2 mRNA expression was analysed, by real-time RT-PCR, in peripheral blood cells isolated from healthy donors. To permit comparison between individuals, absolute copy numbers of NOD2 mRNA were measured during RT-PCR, using cDNA standards, and expressed relative to 10mRNA copies of a validated housekeeping gene HuPO. Lower numbers of PBMCs were harvested from Donors 1 and 3, and these cells were not stimulated with . MV = M, vaccae; H37 = H37RV strain and CPA = Beijing strain

The pathogen recognition sensor, NOD2, is variably expressed in patients with pulmonary tuberculosis-3

<p><b>Copyright information:</b></p><p>Taken from "The pathogen recognition sensor, NOD2, is variably expressed in patients with pulmonary tuberculosis"</p><p>http://www.biomedcentral.com/1471-2334/7/96</p><p>BMC Infectious Diseases 2007;7():96-96.</p><p>Published online 16 Aug 2007</p><p>PMCID:PMC2018706.</p><p></p>rom patients with tuberculosis and matched controls. To permit comparison between individuals, absolute copy numbers of NOD2 (4A) and NOD1 (4B) mRNA were measured during RT-PCR, using cDNA standards, and expressed relative to 10mRNA copies of a validated housekeeping gene HuPO. NOD2 but not NOD1 mRNA levels increased significantly after treatment

The pathogen recognition sensor, NOD2, is variably expressed in patients with pulmonary tuberculosis-2

<p><b>Copyright information:</b></p><p>Taken from "The pathogen recognition sensor, NOD2, is variably expressed in patients with pulmonary tuberculosis"</p><p>http://www.biomedcentral.com/1471-2334/7/96</p><p>BMC Infectious Diseases 2007;7():96-96.</p><p>Published online 16 Aug 2007</p><p>PMCID:PMC2018706.</p><p></p>LR) 2 (p = 0.02) [A] and TLR4 (p = 0.01) [B] in pulmonary leucocytes harvested from patients with tuberculosis. To permit correlations, absolute copy numbers of NOD2 and TLR2, TLR4 and TLR6 mRNA were measured during RT-PCR, using cDNA standards, and expressed relative to 10mRNA copies of a validated housekeeping gene HuPO