Journal: PLoS ONE

Article Title: MyD88/CD40 Genetic Adjuvant Function in Cutaneous Atypical Antigen-Presenting Cells Contributes to DNA Vaccine Immunogenicity

doi: 10.1371/journal.pone.0164547

Figure Lengend Snippet: (A) NIH3T3 or (B) MPEK stable cells lines expressing either GFP alone (neg. control), OVA or MC.OVA were incubated for 60 hours. MC.OVA +rim had 10 nM rim present in the culture. After 60 hours, cells were harvested and MHC-I expression was analyzed by flow cytometry. (C-E) MPEK stable cells surface expression of CD80, CD86, and CD40. MFI values of live cells are reported relative to the mean MFI of negative control cells, transduced with an empty-vector, in each respective sample. (F) CD3 + CD8 + OT-1 or WT T cells were purified by MACs and labeled with CellTrace violet (CTV) dye. Then 5 x 10 4 CD3 + CD8 + CTV dye-labeled OT-1 or WT T cells were incubated with 5 x 10 4 B6-MPEK cells for 60 hours. By flow cytometry, live CTV + cells were analyzed for CTV dye dilution as a measure of cell proliferation. (G) Representative histograms of live CTV + cells. Percentages represent the percent of cells proliferating. n = 3, *p<0.05, One-way ANOVA, Tukey correction for multiple comparisons.

Article Snippet: Cells were then labeled with CellTrace Violet (CTV) cell proliferation dye (ThermoFisher Scientific, Waltham, MA) at either a high (Hi) concentration (5 μM) or low (Lo) concentration (0.5 μM) for 25 minutes at 37°C, per the manufacturer’s recommendations.

Techniques: Expressing, Control, Incubation, Flow Cytometry, Negative Control, Transduction, Plasmid Preparation, Purification, Labeling

Journal: bioRxiv

Article Title: PARK7/DJ-1 promotes pyruvate dehydrogenase activity and maintains Treg homeostasis

doi: 10.1101/2019.12.20.884809

Figure Lengend Snippet: a , Representative histogram overlay of FOXP3 expression, cell proliferation (as labelled by celltrace violet [CTV]) or cell survival as measured by live/dead staining among CD4+ cells differentiated from Th0 cells under the iTreg differentiation condition stimulated for 3 days treated with PDH inhibitor (CPI-613) or control vehicle or without stimulation. b , Percentages of FOXP3 expression among DJ-1 KO or WT CD4+ cells differentiated from Th0 cells under the iTreg differentiation condition stimulated for 3 days. c , Representative flow-cytometry plots of FOXP3 and CTV staining among DJ-1 KO or WT CD4 cells differentiated from Th0 cells under the iTreg differentiation stimulated condition for 3 days. d , Representative flow-cytometry plots of FOXP3 expression among total WT CD4 T cells differentiated from Th0 cells treated with different doses of CPI-613 or control vehicle. e , Representative flow-cytoemtry plots of FOXP3 and CTV staining among total WT CD4 T cells differentiated from Th0 cells treated with different doses of CPI-613 or control vehicle. f , Percentage of living cells under the iTreg differentiation condition for 3 days with different doses of CPI-613 or DCA or control vehicle. g , Representative histogram overlay of FOXP3 expression differentiated from Th0 cells treated with CPI-613 or DCA or control vehicle. The percentages of FOXP3 expressing cells among gated living CD4 T cells were marked. Results represent four ( a, d, e ), three ( f, g ) and two ( b, c ) independent experiments. Data are mean± s.d. The P-values are determined by a two-tailed Student’s t -test. ns or unlabeled, not significant, *P<=0.05, **P<=0.01 and ***P<=0.001.

Article Snippet: Add 1 µl of 5 mM CellTrace™ Violet dye (CTV, C34557, Thermo Fisher Scientific) into 1 ml of PBS and use it to suspend naïve CD4 T cells.

Techniques: Expressing, Staining, Flow Cytometry, Two Tailed Test

Journal: bioRxiv

Article Title: Trypanosoma cruzi amastigotes have a reduced replication rate during chronic stage infections

doi: 10.1101/2020.08.04.236240

Figure Lengend Snippet: CellTrace Violet (CTV) reduces T. cruzi infectivity and inhibits intracellular proliferation. (A) T. cruzi CL-Luc::Scarlet trypomastigotes were incubated with either 5 or 10 µM CTV for 20 minutes and used to infect Vero cell monolayers at a multiplicity of infection of 10:1 (Materials and methods). 18 hours later, infection efficiency was determined by inspecting a total of 2,203 (control), 3,781 (5 µM), and 3,840 (10 µM) Vero cells. At least 300 of these were infected in each case. Each data point corresponds to a randomly acquired image and represents the mean percentage of cells infected. Differences between columns were analysed using a parametric one-way ANOVA with Tukey's post hoc pair wise comparisons. ****p=≤0.0001. (B) Vero cells infected with CTV−ve or CTV+ve trypomastigotes (as above) were incubated for the time periods indicated. The numbers of amastigotes per infected host cell were then determined by analysing >300 infected cells per treatment. Error bars represent the standard deviation from the mean. Data were analysed using a Wilcoxon rank sum test. (C) Images of Vero cells 36 hours after infection with CTV−ve or CTV+ve trypomastigotes. Red, fluorescent T. cruzi amastigotes. Fluorescent parasites containing the CTV tracer dye appear as purple on a red fluorescent background. Size bars=20 µM (D) Images of Vero cells 5 days after infection with trypomastigotes that had been incubated with various concentrations of CTV, as indicated. Blue, intracellular vesicles containing CVT. See also supplementary Video 1. Size bars=20 µM, except where indicated; *=50 µM.

Article Snippet: T. cruzi trypomastigotes were isolated by centrifugation and allowed to recover for 2 hours at 37ºC in high-glucose DMEM medium with 10% FBS, and then labelled with the CellTrace Violet (CTV) fluorescent dye (Thermo Fisher Scientific) according to the manufacturer’s protocol.

Techniques: Infection, Incubation, Standard Deviation