mouse treg cell staining kit  (Multi Sciences (Lianke) Biotech Co Ltd)

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Exogenous acetate mitigates later enhanced allergic airway inflammation in a menopausal mouse model

doi: 10.3389/fcimb.2025.1543822

Figure Lengend Snippet: Preventive acetate treatment enhances the percentage of lung Treg cells in re-challenged OVx allergic mice. Acetate was administered 15 days prior to sensitization and continued until one day before the first challenge (day 20).Ten days after the last challenge, the ovaries were removed. Ten days after ovariectomy (OVx), animals underwent rechallenge. Twenty-four hours after rechallenge, cells from the BAL fluid were analyzed by flow cytometry and stained for CD4-APC, CD25-PE, and FoxP3-FITC. (A) Total cells, (B) Lymphocytes, (C) Percentage of Treg cells and (D) Number of Treg cells. All the results are expressed as the means ± SEMs (n = 6-7 in all the groups). Statistical significance was determined by one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test. *P < 0.05, **P < 0.01.

Article Snippet: Mouse Treg cells were collected from the BAL fluid and analyzed for CD4/CD25/Foxp3 expression via a mouse Treg cell staining kit (eBioscience).

Techniques: Flow Cytometry, Staining

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Exogenous acetate mitigates later enhanced allergic airway inflammation in a menopausal mouse model

doi: 10.3389/fcimb.2025.1543822

Figure Lengend Snippet: Schematic representation of the effect of acetate treatment on the lung and gut of rechallenged OVx allergic mice ( Peacock et al., 2023 ). Acetate was administered 15 days prior to sensitization and continued until one day before the first challenge (day 20) ( Colombo et al., 2019 ). Ten days after the last challenge, the ovaries were removed ( Chowdhury et al., 2021 ). Ten days after ovariectomy (OVx), the animals underwent rechallenge. Twenty-four hours after rechallenge, cells from the BAL fluid and gut were analyzed. Mice treated with acetate showed a significant reduction in cell infiltration, mucus production, IgE, and cytokine levels, as well as an increased percentage of Treg cells in the airways compared to mice treated with saline. Additionally, acetate treatment increased the expression of MUC2 and OCLN and decreased mucus intensity in the gut. Created in BioRender. De oliveira ribeiro, F. (2025) https://BioRender.com/p79j710 .

Article Snippet: Mouse Treg cells were collected from the BAL fluid and analyzed for CD4/CD25/Foxp3 expression via a mouse Treg cell staining kit (eBioscience).

Techniques: Saline, Expressing

Journal: bioRxiv

Article Title: Tissue-resident uterine regulatory T cells support fetal growth

doi: 10.1101/2023.09.07.554921

Figure Lengend Snippet: A) Gating strategy to identify Treg cells among live CD45 + cells in the uterus and udLNs of non-pregnant Foxp3 eGFP reporter mice (C57BL6 background). B) Percentages of Foxp3 eGFP -expressing cells among CD4 + T cells in the indicated organs (n=12). C) Histograms (right) show the expression levels of CD25 as measured by the mean fluorescence intensity (MFI) among Tregs from Uterus and uterus-draining lymph nodes (udLN) (n=12). D) Histogram showing CD25 expression in Tregs from perfused uterus (red) and udLN (blue) (right panel). Numbers indicates Mean Fluorescence Index. E) Memory populations frequencies among the resident uTregs and blood circulating Tregs. Resident Tregs from perfused uterus are effector memory CD45 + CD3 + CD4 + CD44 + CD62L - CD25 low Foxp3 + cells. (n=4-12 females). ( F-G ): Histograms showing that Tregs from perfused uterus express activation and tissue-resident markers: F) Frequency of IL33R and CD103 expressing Tregs. (n=4-12 females). G) uTreg expression of CTLA-4 and Helios compared to Tregs from udLNs. (n=4-12 females). H) Parabiosis experiment showing the migratory capacity of T cell populations in the uterus and the secondary lymphoid organs. T cells have a high residency in uterine tissue. Foxp3 eGFPIRES BALB/cJ Thy1.1 mice were joined to BALB/cJ Thy1.2. Gating strategy of CD4 + T cells in parabiosis: host and donor cells were identified with Thy1.1 (CD90.1) and Thy1.2 (CD90.2) congenic markers among both Foxp3-GFP BALB/cJ Thy1.1 and BALB/cJ Thy1.2 mice. (n=8 parabionts for a total of 16 mice) ( *p<0.05, **p<0.009, ***p<0.0009 ). I) The percentages of Thy1.1 and Thy1.2 among CD4 + Foxp3 - and CD4 + Foxp3 + T cells in the uterus, udLNs, parabiosis dLNs (pbdLNs) and non-dLNs (ndLNs) were used to calculate the host-to-donor ratio in each parabiont 14 days after surgery. The dotted horizontal line represents a 1:1 host-to-donor ratio of complete chimerism (n=8 parabionts for a total of 16 mice) ( *p<0.05, **p<0.009, ***p<0.0009 ). EM: effector memory, CM: central memory, Eff: effector, Nv: naïve.

Article Snippet: Intracellular staining was performed using the FOXP3 mouse Treg cell staining kit (eBiosciences 00552300).

Techniques: Expressing, Fluorescence, Activation Assay

Journal: bioRxiv

Article Title: Tissue-resident uterine regulatory T cells support fetal growth

doi: 10.1101/2023.09.07.554921

Figure Lengend Snippet: ( A-B ) uTregs proliferate: A) 70% of uTregs are Ki67 + , percentages of Ki67 + Foxp3 + cells in non-perfused (n=5, left panel) and perfused (n=4) uterus and udLNs (right panel) from C57BL6 mice. B) Histogram representing Edu-labeled cells among uTregs and udLN Tregs (left panel). Percentages of Edu + Tregs and CD4conv cells in the uterus, udLNs, ndLNs and spleen of non-pregnant animals (n=4, *p<0.05). C) Log2 of quantile normalized expression for all the genes belonging to the interleukin, tumor necrosis factor (TNF) and interferon families and their corresponding receptors from the HGCN database within the GSE68454 dataset. IL15, TGF-β, IL33 and TNF are present in the non-pregnant uterus. D) Relative expression levels (1 / ΔCt) of T cells related to cytokines in the uterus and udLNs. Differences among the highly expressed cytokines—IL15 TGF-β and IL33—and poorly expressed cytokines—IL-4, IL2, IL-10, IFNɣ and TNFα—are significant in the uterus ( p=0.0286 ) and udLNs ( p=0.0079 ). IL2 levels are lower in uterine tissue than in lymphoid tissue ( p=0.0159 ) (n=4). ( E-F ) IL2 is scarce in the non-pregnant uterus: E) Log2 fold change in the relative expression of uterine cytokines vs. IL2 by qPCR ( p=0.0286 ) (n=4). F) Cytokine IL2 concentration measured by ELISA IL2 from the non-pregnant uterus, udLNs and ndLNs (n=4). G) Uterine Treg cells in C57BL/6 mice infected with AAV-IL2 regain CD25 expression. Dot plots of Foxp3 and CD25 among CD4 + cells in the uterus and udLNs after AAV-IL2 treatment compared to the AAV-Luc control. H) Percentages of CD25 + cells among Tregs ( p=0.0043 ) after AAV-IL2 treatment (n=6 representative of 2 independent experiments). I) IL33R, J) GARP and K) IL15Rα are expressed by uTregs. Histograms showing IL33R + , GARP + and IL15Rα + cells among CD45 - (violet), CD45 + CD3 - (blue), CD3 + Foxp3 - (green), Foxp3 + (red) uterine (upper panels) and udLN (lower panels) populations from C57BL/6 mice.

Article Snippet: Intracellular staining was performed using the FOXP3 mouse Treg cell staining kit (eBiosciences 00552300).

Techniques: Labeling, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Infection

Journal: bioRxiv

Article Title: Tissue-resident uterine regulatory T cells support fetal growth

doi: 10.1101/2023.09.07.554921

Figure Lengend Snippet: RNA-Seq analysis from purified CD25-uTregs, uterine CD4 + Foxp3 - (CD4conv) T cells and udLN CD25 + Tregs (n=3). A ) PCA of transcriptional profiles distinguishing NP uTregs, NP uterine CD4conv and NP udLN Tregs. B) Volcano plot showing genes differentially expressed between NP uTregs and NP uterine CD4conv (left) and between NP uTregs and NP udLN Tregs (right). C ) Network representation of pathway enrichment analysis from the list of pathways statistically differentially expressed between NP uTregs and the indicated groups. Each node in the representation corresponds to a pathway, and nodes are connected if the lists of genes in the associated pathways are overlapping. Nodes are colored using a color-gradient scale ranging from blue (<0) to red (>0) according to the difference of mean expressions between the conditions. Clusters of pathways are distinguished from each other by their grey-colored area. D ) Details of the enrichment of biological pathways related to observed ECM, FGF signaling, and vascular system clusters in the enrichment network shown in the NP uTregs vs. NP udLN Tregs comparison. Expression score for each pathway found to be statistically differently expressed in the different individual are represented using a color-gradient scale ranging from yellow (low) to blue (high). E ) Heatmap of comparison between differentially expressed genes vs. bulk lymph-nodes cells (LN) of Tregs from NP or E6-E8 uterus, and Tregs from visceral adipose tissue (VAT), injured muscle, colonic lamina propria and lung IL18 + Tregs. Blue boxes indicate up-regulation of the gene in the given condition, yellow boxes indicate down-regulation. Grey boxes indicate NA values.

Article Snippet: Intracellular staining was performed using the FOXP3 mouse Treg cell staining kit (eBiosciences 00552300).

Techniques: RNA Sequencing Assay, Purification, Comparison, Expressing

Journal: bioRxiv

Article Title: Tissue-resident uterine regulatory T cells support fetal growth

doi: 10.1101/2023.09.07.554921

Figure Lengend Snippet: A) uTregs cell numbers increase during the allogeneic pregnancy of female Foxp3 eGFP (C57BL/6) mice mated with BALB/C males. Histograms show the numbers of Tregs ( p<0.01 ) among total uterine cells. B) Tregs present an activated memory phenotype at E8 and E12 days of pregnancy. Percentages of memory populations among uTregs. (n=3 to 5). C) CD25 is modulated among Tregs during pregnancy. Line bar graphs show the expression levels in MFI of CD25 in Tregs, uterus (red line) and udLN (black line) tissues. Data from NP, E8, and E12 of pregnancy. ( *p<0.05, **p<0.01, ***p<0.001 ) (n= 3 to 16 mice representative of 2 different experiments). (D-G): Percentages of D) CD103 + E) CTLA-4 + , F) Ki67 + and G) Helios + cells among uterine or udLN Tregs. Data from NP, E8 and E12 uterus (red line) and udLN (black line) tissues ( *p<0.05, **p<0.01, ***p<0.001 ) (n= 3 to 16 mice representative of 2 different experiments). H) Adoptive transfer of Thy1.1 CD4 + GFP - cells into C57BL/6 females BALB/c mated. Gating strategy showing the induction of uTregs determined by the expression of Foxp3-GFP. pTregs appear at E13 of mid-gestation. Data representative of 3 independent experiments.

Article Snippet: Intracellular staining was performed using the FOXP3 mouse Treg cell staining kit (eBiosciences 00552300).

Techniques: Expressing, Adoptive Transfer Assay

Journal: bioRxiv

Article Title: Tissue-resident uterine regulatory T cells support fetal growth

doi: 10.1101/2023.09.07.554921

Figure Lengend Snippet: A) Principal component analysis (PCA) based on normalized gene expression showing the coordinated dynamics of hypoxia and vasculogenesis signatures with uTreg DEGs. PCA accurately separates non-pregnant (NP – E4) mice and those in early (at E6 and E8, after embryo implantation) or late allogeneic pregnancy (at E10, E11 and E12, C75BL/6 females mated with BALB/c males), (n = 4 to 6). B) Regularized generalized canonical correlation (RGCC) analysis for multiblock data sets representing the first component from each PCA of uTregs, vasculogenesis and hypoxia signatures from the same samples as (A). The numbers on the side are the correlation coefficients between the first components extracted from each signature. The colors and ellipses are related to the pregnancy time points and indicate the power of each component to discriminate between the different stages of pregnancy. C) Differential transcriptional analysis of total uteri after Treg depletion in the Foxp3 DTR mouse line. Transcriptomic module analysis of Treg sufficient or Treg depleted total uteri (n = 4 per group, see the Methods section for the depletion protocol). Modules shows gene clusters from molecular signatures related to blood vessel formation and development, and extracellular matrix functions. Cluster labels represent MSigDB molecular signatures from C2 (curated gene sets) and C5 (GO gene sets). The average expression over all genes in each module is shown per sample (rows) colored from red (over-expressed) to blue (under-expressed). Module expression correlates with sample status (DT vs. PBS) (chi-square test p-value < 5e -2 ) and is significantly different between the groups (t-test p-value < 1e -2 ). D) Enrichment scores of vascular system-associated pathways identified in the different lists of differentially expressed genes. Significant items are represented with a star and are colored based on the directionality of the dysregulation. E) Confluence assay during 90h co-culture between uTregs (red), uDLN (blue) or non-DLN (green); and with mouse primary uterine microvascular endothelial cells (muMECs). Data represent 2 different experiments, 4 points, 10 E6-E8 pregnant BALB/c-mated or NP C57BL/6 females per point. Significant differences are denoted by **: P < 0.01, ***: P < 0.001 and ****: P < 0.0001. F) Conceptus area micrography of normal gravid uterus at E8 or gravid uterus with fetuses presenting intrauterine growth restriction (IUGR), respectively from PBS or DT-treated FoxP3 DTR mice (C57BL/6 background mated with BALB/c males) (scale bar = 1cm, n=5). G) Scatter dot plot of conceptus area at E8 from WT (C57BL/6) or Foxp3 DTR mice that received PBS or DT every 2 days from E1,5, or DT every 2 days from E1,5 plus 5-9 × 10 6 T reg cells from WT mice. Each littermate is distinguishable by a distinct color and shape (Foxp3 DTR + PBS: n = 6 littermates; Foxp3 DTR + DT: n= 8; WT + DT: n = 5; WT + PBS: n = 2; Foxp3 DTR + DT + Tregs: n = 5). Data represent 198 embryos in total from C57BL/6 or FoxP3 DTR mice mated with BALB/c males (7 experiments). Red lines represent mean and standard deviation. A Student’s t test was used for statistical analysis. ns: non-significant; ****: P < 0.0001.

Article Snippet: Intracellular staining was performed using the FOXP3 mouse Treg cell staining kit (eBiosciences 00552300).

Techniques: Expressing, Co-Culture Assay, Standard Deviation