Journal: Cell Reports Methods

Article Title: A genetic tool for the longitudinal study of a subset of post-inflammatory reactive astrocytes

doi: 10.1016/j.crmeth.2022.100276

Figure Lengend Snippet: Expression of Lcn2CreERT2 following repeated low-dose LPS treatment (A) Immunostaining for tdTomato in Lcn2CreERT2 ; Ai9 brains. Animals were given one of the following four treatments for 5 consecutive days: Saline and corn oil, saline and tamoxifen, LPS and corn oil, LPS and tamoxifen. Sections were counterstained with DAPI. (B) Quantification of total number of red cells/section from animals in (A). ∗∗∗p < 0.001 ANOVA and Tukey’s post hoc test, n = 5 animals per treatment. (C) Immunostaining of brains from saline- (left) or LPS-treated (right) Lcn2CreERT2 ; Ai9 animals for tdTomato and the astrocytic marker GFAP (top), the endothelial and microglial marker lectin (second row), the neuronal marker NeuN (third row), and the microglial marker Iba1 (bottom). Sections were counterstained with DAPI. White arrowheads indicate co-positive cells for tdTomato and the panel maker. (D) Quantification of co-positive cells from (C). (E) RNAscope for the astrocytic marker Slc1a3 (top) and the endothelial marker Pecam1 (bottom) on brains from LPS- and saline-treated Lcn2CreERT2 ; Ai9 animals. Sections were immunostained for tdTomato and counterstained with DAPI. (F) Quantification of cells co-positive for tdTomato and RNA markers described in (E). Scale bars, (A) 250 μm, (C) 50 μm, (E) 25 μm. Error bars ± SEM. See also Figures S1 and .

Article Snippet: RNAScope Multiplex Fluorescent Detection kit v2 , Advanced Cell Diagnostics , 323110.

Techniques: Expressing, Immunostaining, Saline, Marker, RNAscope

Journal: Cell Reports Methods

Article Title: A genetic tool for the longitudinal study of a subset of post-inflammatory reactive astrocytes

doi: 10.1016/j.crmeth.2022.100276

Figure Lengend Snippet: Astrocytes that were initially reactive remain in the brain after resolution of inflammation (A) Lcn2CreERT2 ; Ai9 brain sections immunostained for tdTomato and counterstained with DAPI. Animals were given tamoxifen and saline or LPS and collected at either 24 h or 1 month after the last treatment. (B) Quantification of tdTomato-positive cells in the thalamic region. p = 0.38, ANOVA followed by Tukey’s HSD post hoc, n = 5 animals. (C) RNAScope on Lcn2CreERT2 ; Ai9 brain sections for the astrocyte marker Aldh1l1 24 h and 1 month after low-dose LPS treatment, followed by immunostaining for tdTomato and counterstaining with DAPI. (D) Quantification of Aldh1l1 and tdTomato co-positive cells in (C). p = 0.16, paired t test. n = 18 pooled sections across three animals. (E) Co-labeling of tdTomato and EdU-positive astrocytes in Lcn2CreERT2 ; Ai9 animals after LPS treatment. (F) High magnification image of labeled astrocytes and EdU-positive cells at 1 month. (G) Quantification of tdTomato and EdU co-positive astrocytes at 24 h, 7 days, and 1 month following LPS treatment. Error bars ± SEM. Scale bars, (A) 400 μm, (C, E) 200 μm, (F) 20 μm. See also Figure S4 .

Article Snippet: RNAScope Multiplex Fluorescent Detection kit v2 , Advanced Cell Diagnostics , 323110.

Techniques: Saline, RNAscope, Marker, Immunostaining, Labeling

Journal: Cell Reports Methods

Article Title: A genetic tool for the longitudinal study of a subset of post-inflammatory reactive astrocytes

doi: 10.1016/j.crmeth.2022.100276

Figure Lengend Snippet: Reactive marker expression decreases 1 month after inflammation (A and B) Analysis of reactive marker expression within tdTomato-positive cells. RNAScope for brain sections 24 h after LPS, or 1 month after LPS treatment to detect expression of the reactive markers Lcn2 (A) and GFAP (B), followed by immunostaining for tdTomato and counterstaining with DAPI. RNAScope for reactive markers Lcn2 (A) and GFAP (B) was quantified by looking at both number of fluorescent puncta (A, B, top panels) and integrated fluorescent density (A, B, bottom panels). ∗∗∗p < 0.001, paired t test. n = 29 astrocytes pooled from three animals. Error bars ± SEM. Scale bars, 50 μm. See also Figure S5 .

Article Snippet: RNAScope Multiplex Fluorescent Detection kit v2 , Advanced Cell Diagnostics , 323110.

Techniques: Marker, Expressing, RNAscope, Immunostaining

Journal: Cell Reports Methods

Article Title: A genetic tool for the longitudinal study of a subset of post-inflammatory reactive astrocytes

doi: 10.1016/j.crmeth.2022.100276

Figure Lengend Snippet: Reactive markers return to baseline levels in astrocytes that were initially reactive (A–C) RNAScope staining (green) for Slc1a2 (A), Slc1a3 (B), and Lrrc8a (C) in Lcn2CreERT2 ; Ai9 animals treated with saline or LPS. Sections were immunostained for tdTomato and counterstained with DAPI. Bottom: quantification of RNAScope puncta per tdTomato + astrocyte. ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, ANOVA followed by Tukey’s honest significant difference post hoc test. n = 30 pooled astrocytes across three animals. Error bars ± SEM. Scale bars, 20 μm.

Article Snippet: RNAScope Multiplex Fluorescent Detection kit v2 , Advanced Cell Diagnostics , 323110.

Techniques: RNAscope, Staining, Saline

Journal: Cell Reports Methods

Article Title: A genetic tool for the longitudinal study of a subset of post-inflammatory reactive astrocytes

doi: 10.1016/j.crmeth.2022.100276

Figure Lengend Snippet:

Article Snippet: RNAScope Multiplex Fluorescent Detection kit v2 , Advanced Cell Diagnostics , 323110.

Techniques: Virus, Recombinant, Plasmid Preparation, RNAscope, Multiplex Assay

Journal:

Article Title: Colonic epithelial cell mediated suppression of CD4 T cell activation

doi: 10.1136/gut.2003.029967

Figure Lengend Snippet: Colonic epithelial cells (CEC) inhibit the response of T cells to conventional mitogens. Splenic CD4 T cells (A, B, D, F, G) or colonic lamina propria CD4 T cells (C, E) were cultured with freshly isolated C57BL/6 CEC or macrophages (Mφ) in the presence of mitogenic CD3 antibody for 72 hours prior to flow cytometric analysis for expression of CD25 on splenic (A) and lamina propria T cells (C), CD45RB on splenic CD4 T cells (B), cell cycle progression of splenic T cells (D) and lamina propria T cells (E), and cytokine production of splenic T cells using cytokine bead assays (F, G). In (A), (B), and (C), the filled in and solid line profiles represent levels of antibody staining of T cells after coculture with Mφ and CEC, respectively, and the broken lines represent the isotype matched control antibodies. (D, E) Proportion of splenic and lamina propria CD4 T cells in the G1, S, and G2 phases of the cell cycle. (F, G) Cytokines produced by splenic T cells after culture with CEC and Mφ in the presence of mitogen, respectively. Results are representative of those obtained from at least six independent experiments. IFN-γ, interferon γ; IL, interleukin; TNF-α, tumour necrosis factor α.

Article Snippet: Cytokine multiplex bead assay kits (Becton Dickinson) were used to measure levels of interferon γ (IFN-γ), tumour necrosis factor α (TNF-α), interleukin (IL)-5, IL-4, and IL-10 in CEC:T and Mφ:T cell conditioned media.

Techniques: Cell Culture, Isolation, Expressing, Staining, Control, Produced

Journal:

Article Title: Colonic epithelial cell mediated suppression of CD4 T cell activation

doi: 10.1136/gut.2003.029967

Figure Lengend Snippet: Responses of antigen specific T cells are inhibited by colonic epithelial cells (CEC). I-Ad-restricted DO11.10 CD4 T cells were cocultured with OVA primed DO11.10 CEC or macrophages (Mφ) in the presence or absence of OVA for 72 hours prior to flow cytometric analysis of T cells for expression of CD25 (A), CD45RB (B), cell cycle progression (C), and cytokine production (D). (A, B) Cells cultured in the absence or presence of OVA: **p<0.005 (A) and **p<0.005 (B) comparing CD4 T cell activation in the presence of BALB/C (I-Ad) CEC or Mφ. (C) Proportion of CD4 T cells in the G1, S, and G2 phases of the cell cycle, respectively, with the plus and minus symbols referring to the presence or absence of OVA in the coculture. (D) Cytokine production by CD4 T cells cocultured with CEC and Mφ, respectively. Results are representative of those obtained from at least eight independent experiments. IFN-γ, interferon γ; IL, interleukin; TNF-α, tumour necrosis factor α. (E) C57BL/6 (I-Ab) CEC can present OVA to an OVA specific I-Ab restricted CD4 T cell hybridoma KZO. Cultured CEC were pulsed with OVA, washed, and incubated for 24 hours with KZO, which had been transfected with an NFAT-lacZ reporter gene construct. T cell hybridoma activation and lacZ activity were determined spectrophotometrically using the chromogenic substrate and measuring absorbance at 595 nm (OD 595).

Article Snippet: Cytokine multiplex bead assay kits (Becton Dickinson) were used to measure levels of interferon γ (IFN-γ), tumour necrosis factor α (TNF-α), interleukin (IL)-5, IL-4, and IL-10 in CEC:T and Mφ:T cell conditioned media.

Techniques: Expressing, Cell Culture, Activation Assay, Incubation, Transfection, Construct, Activity Assay

Journal: Journal of Clinical Virology

Article Title: High-sensitivity SARS-CoV-2 group testing by digital PCR among symptomatic patients in hospital settings

doi: 10.1016/j.jcv.2021.104895

Figure Lengend Snippet: Distribution of the samples identified as positive by the routine RT-PCR method (Cobas® SARS-CoV-2 assay) in the groups of 8, 16 and 32 and corresponding RT-dPCR detection of SARS-CoV-2 in the groups.

Article Snippet: Three different group sizes were investigated using a commercially available digital PCR assay, the COVID-19 Multiplex Digital PCR Detection Kit (Stilla Technologies, France/Apexbio, China).

Techniques: Digital PCR

Journal: Journal of Clinical Virology

Article Title: High-sensitivity SARS-CoV-2 group testing by digital PCR among symptomatic patients in hospital settings

doi: 10.1016/j.jcv.2021.104895

Figure Lengend Snippet: Confusion matrix for P8 , 16 and 32 groups of samples .

Article Snippet: Three different group sizes were investigated using a commercially available digital PCR assay, the COVID-19 Multiplex Digital PCR Detection Kit (Stilla Technologies, France/Apexbio, China).

Techniques:

Journal: Journal of Clinical Virology

Article Title: High-sensitivity SARS-CoV-2 group testing by digital PCR among symptomatic patients in hospital settings

doi: 10.1016/j.jcv.2021.104895

Figure Lengend Snippet: Detailed results of confirmatory testing for COBAS-/dPCR+ discordances by individual reassessment using both RT-dPCR and RT-PCR (Altona and Cobas®). NT = “Not tested”. ND= “Not detected”.

Article Snippet: Three different group sizes were investigated using a commercially available digital PCR assay, the COVID-19 Multiplex Digital PCR Detection Kit (Stilla Technologies, France/Apexbio, China).

Techniques: