pe cy7 rat anti-mouse cd4  (Thermo Fisher)

Journal: Physiological Reports

Article Title: T cell‐expressed Ift88 is required for proper thymocyte differentiation in mice

doi: 10.14814/phy2.70120

Figure Lengend Snippet: Loss of Ift88 in thymic epithelial cells does not impact T cell development. (a) Whole mount image of SSTR3:GFP cilia reporter mice co‐stained with acetylated alpha tubulin and keratin 5 using a 60× objective (b, c) Thymus (b) and spleen (c) weight in Foxn1 cre+ Ift88 f/f or control mice at 8 weeks of age. (d, e) Representative FACS plots showing DN1‐DN4 thymocytes (d), and CD4+/CD8+ DP, CD4+ SP, and CD8+ SP thymocytes (e) in 8 week old Foxn1 cre+ Ift88 f/f or control mice. (f–h) Quantification of DN1‐DN4 thymocytes (f), DP thymocytes (g), and CD4+ and CD8+ SP thymocytes (h) in Foxn1 cre+ Ift88 f/f or control mice. (i) Representative FACS plots showing CD4+ and CD8+ T cells in the spleen of Foxn1 cre+ Ift88 f/f or control mice at 8 weeks of age. (j) Quantification of CD4+ and CD8+ spleenocytes in Foxn1 cre+ Ift88 f/f or control mice. For each quantification (except spleen weight), N = 10 mice per group (Controls: 6M, 4F; Foxn1 cre+ Ift88 f/f : 7M, 3F). Two‐way ANOVA.

Article Snippet: Primary antibodies were as follows: APC rat anti‐mouse CD25 (Catalog#: 101910, clone 3C7, BioLegend), PE rat anti‐mouse CD3 (Catalog#: 12–0031‐82, clone 145–2 c11, eBioscience), PE Cy7 rat anti‐mouse CD4 (Catalog#: 25–0041‐82, clone GK1.5, Invitrogen), Percpcy5.5 rat anti‐mouse CD8 (Catalog #: 45–0081‐82, clone 53–6.7, Invitrogen), BV786 rat anti‐mouse CD44 (Catalog#: 563736, clone IM7, BD Bioscience), BV605 hamster anti‐mouse TCRβ (Catalog#: 109241, H57‐ APC 597, BioLegend), FITC rat anti‐mouse CD45 (Catalog#: 2023‐08‐08, Clone 30‐F11, ThermoFisher Scientific), and Fixable Aqua Dead Cell Stain (Catalog#: L34957, Invitrogen).

Techniques: Staining, Control

Journal: Physiological Reports

Article Title: T cell‐expressed Ift88 is required for proper thymocyte differentiation in mice

doi: 10.14814/phy2.70120

Figure Lengend Snippet: Loss of Ift88 in T cells prevents DP to SP thymocyte transition. (a, b) FACS plots showing DN1‐DN4 thymocytes (a) and CD4+/CD8+ DP, CD4+ SP, and CD8+ SP thymocytes (b) that are TdT+ (left) and GFP+ (right) 3 weeks post tamoxifen induction in CaggCre ERT2+ Ift88 f/f mTmG mice. (c) Quantification of the ratio of GFP+ to TdT+ thymocytes during thymocyte development. Data is shown as the percentage of parent cells that express GFP or TdT. (d–f) Quantification of total DN1‐DN4 thymocytes (d), DP thymocytes (e), and CD4+ and CD8+ SP thymocytes (f) in CaggCre ERT2+ Ift88 f/f mTmG or control mice. For each graph, N = 5 mice (all male). Two‐way ANOVA.

Article Snippet: Primary antibodies were as follows: APC rat anti‐mouse CD25 (Catalog#: 101910, clone 3C7, BioLegend), PE rat anti‐mouse CD3 (Catalog#: 12–0031‐82, clone 145–2 c11, eBioscience), PE Cy7 rat anti‐mouse CD4 (Catalog#: 25–0041‐82, clone GK1.5, Invitrogen), Percpcy5.5 rat anti‐mouse CD8 (Catalog #: 45–0081‐82, clone 53–6.7, Invitrogen), BV786 rat anti‐mouse CD44 (Catalog#: 563736, clone IM7, BD Bioscience), BV605 hamster anti‐mouse TCRβ (Catalog#: 109241, H57‐ APC 597, BioLegend), FITC rat anti‐mouse CD45 (Catalog#: 2023‐08‐08, Clone 30‐F11, ThermoFisher Scientific), and Fixable Aqua Dead Cell Stain (Catalog#: L34957, Invitrogen).

Techniques: Control

Journal: Physiological Reports

Article Title: T cell‐expressed Ift88 is required for proper thymocyte differentiation in mice

doi: 10.14814/phy2.70120

Figure Lengend Snippet: The ratio of GFP+ to TdT+ T cells in the spleen and kidney is substantially reduced. (a, b) FACS plots showing TdT+ and GFP+ CD4+ and CD8+ T cells in the spleen (a) and kidney (b) 3 weeks post‐tamoxifen induction in CaggCre ERT2+ Ift88 f/f mTmG mice. (c, d) Quantification of the ratio of GFP+ to TdT+ cells in the spleen (c) and kidney (d) 3 weeks post‐tamoxifen induction. (e, f) Quantification of total CD4+ and CD8+ T cells in the spleen (e) and kidney (f) 3 weeks post‐tamoxifen induction. N = 5 mice (all male). Two‐way ANOVA.

Article Snippet: Primary antibodies were as follows: APC rat anti‐mouse CD25 (Catalog#: 101910, clone 3C7, BioLegend), PE rat anti‐mouse CD3 (Catalog#: 12–0031‐82, clone 145–2 c11, eBioscience), PE Cy7 rat anti‐mouse CD4 (Catalog#: 25–0041‐82, clone GK1.5, Invitrogen), Percpcy5.5 rat anti‐mouse CD8 (Catalog #: 45–0081‐82, clone 53–6.7, Invitrogen), BV786 rat anti‐mouse CD44 (Catalog#: 563736, clone IM7, BD Bioscience), BV605 hamster anti‐mouse TCRβ (Catalog#: 109241, H57‐ APC 597, BioLegend), FITC rat anti‐mouse CD45 (Catalog#: 2023‐08‐08, Clone 30‐F11, ThermoFisher Scientific), and Fixable Aqua Dead Cell Stain (Catalog#: L34957, Invitrogen).

Techniques:

Journal: Communications Biology

Article Title: Vaccine-elicited IL-1R signaling results in Th17 TRM-mediated immunity

doi: 10.1038/s42003-024-06138-0

Figure Lengend Snippet: a Representative UMAP plot of Il1r1 expression in CD4+ cells from naïve spleens and OmpX+LTA1 immunized lungs. b The Log2 expression levels of Il1r1 from lung CD4+ T cells (lung) and naïve splenic CD4+ T cells (spleen) shown in a . c Representative flow cytometry histograms showing IL-1R1 on CD4+ T cells (CD45 + , CD19 - , CD3 + , CD4 + ) from single cell lung suspensions of PBS treated and OmpX+LTA1 vaccinated mice. Concentrations of ( d ) GM-CSF, ( e ) IL-17A, ( f ) IL-6, ( g ) IL-27, ( h ) IL-10, ( i ) MCP-1, ( j ) IL-12p70, ( k ) IFN-β, ( l ) IFNγ, ( m ) TNF, ( n ) IL-1α, and ( o ) IL-1β in the serum, BAL fluid, and lung homogenate of immunized and unimmunized mice. Data are presented as the mean +/− SEM, n = 3 mice per group. Data were analyzed using the unpaired T test.

Article Snippet: Antibodies used for blocking and staining are as follows: Rat Anti-Mouse CD16/CD32 Fc Block (clone 2.4G2, BD Biosciences, Cat #553141), PE-Cy7 Rat Anti-Mouse CD4 (clone RM4-5, BD Biosciences, Cat #561099), APC rat anti-mouse CD3e (clone 17A2, BioLegend, Cat #100236), PE-Cy5 hamster anti-mouse TCRβ (clone H57-597, BD Biosciences, Cat #553173), FITC rat anti-mouse IL-17A (clone TC11-18H10.1, BioLegend, Cat #506907), Brilliant Violet 421 rat anti-mouse IFNγ (clone XMG1.2, BioLegend, Cat #505829), PE rat anti-mouse B220 (clone RA3-6B2, BioLegend, Cat #103207).

Techniques: Expressing, Flow Cytometry

Journal: Communications Biology

Article Title: Vaccine-elicited IL-1R signaling results in Th17 TRM-mediated immunity

doi: 10.1038/s42003-024-06138-0

Figure Lengend Snippet: a CD4 T cell numbers, b B cell numbers, and c Th17 cell numbers out of 50,000 events from the lungs of vaccinated and unvaccinated wild-type and Il1r1 −/− mice as measured in flow cytometry ( n = 7 mice per group). d Representative dot plots for determining lung Th17 cells (CD3 + , CD4 + , TCR-β + , IL-17A + ) in immunized and unimmunized wildtype and Il1r1 −/− mice. e Median fluorescent intensity of FITC stained IL-17A in vaccinated and control mice ( n = 7 mice per group). f IgA titers specific to heat killed K. pneumoniae obtained from the supernatant of homogenized lungs in each group ( n = 5 mice per group). g Area under the curve analysis of data displayed in f . Log transformed bacterial burdens in the ( h ) lungs and ( i ) spleen 24 h post-challenge in each group were measured in a cfu plating assay ( n = 9 mice, IL-1R1 −/− PBS; n = 11 mice, WT PBS; n = 13 mice, IL-1R1 −/− Imm and WT Imm). All data are represented as mean +/− SEM. Statistical differences were determined using a one-way ANOVA followed by Tukey’s multiple comparison test. Imm immunized.

Article Snippet: Antibodies used for blocking and staining are as follows: Rat Anti-Mouse CD16/CD32 Fc Block (clone 2.4G2, BD Biosciences, Cat #553141), PE-Cy7 Rat Anti-Mouse CD4 (clone RM4-5, BD Biosciences, Cat #561099), APC rat anti-mouse CD3e (clone 17A2, BioLegend, Cat #100236), PE-Cy5 hamster anti-mouse TCRβ (clone H57-597, BD Biosciences, Cat #553173), FITC rat anti-mouse IL-17A (clone TC11-18H10.1, BioLegend, Cat #506907), Brilliant Violet 421 rat anti-mouse IFNγ (clone XMG1.2, BioLegend, Cat #505829), PE rat anti-mouse B220 (clone RA3-6B2, BioLegend, Cat #103207).

Techniques: Flow Cytometry, Staining, Transformation Assay, Comparison

Journal: Communications Biology

Article Title: Vaccine-elicited IL-1R signaling results in Th17 TRM-mediated immunity

doi: 10.1038/s42003-024-06138-0

Figure Lengend Snippet: a CD4 T cell numbers, b B cell numbers, and c Th17 cell numbers out of 50,000 events from the lungs of unvaccinated WT or vaccinated WT and Casp1 −/− mice, as measured in flow cytometry. d Representative dot plots of Th17 cells (CD3 + , CD4 + , TCR-β + , IL-17A + ) in each group. Log transformed bacterial burdens in the ( e ) lungs and ( f ) spleens 24 h post-challenge as determined using cfu plating assays. Data are presented as mean +/− SEM ( n = 10 mice, Casp1 −/− Imm; n = 8 mice, WT Imm; n = 6 mice, WT PBS). Statistical differences were determined using a one-way ANOVA followed by Tukey’s multiple comparison test. Imm immunized.

Article Snippet: Antibodies used for blocking and staining are as follows: Rat Anti-Mouse CD16/CD32 Fc Block (clone 2.4G2, BD Biosciences, Cat #553141), PE-Cy7 Rat Anti-Mouse CD4 (clone RM4-5, BD Biosciences, Cat #561099), APC rat anti-mouse CD3e (clone 17A2, BioLegend, Cat #100236), PE-Cy5 hamster anti-mouse TCRβ (clone H57-597, BD Biosciences, Cat #553173), FITC rat anti-mouse IL-17A (clone TC11-18H10.1, BioLegend, Cat #506907), Brilliant Violet 421 rat anti-mouse IFNγ (clone XMG1.2, BioLegend, Cat #505829), PE rat anti-mouse B220 (clone RA3-6B2, BioLegend, Cat #103207).

Techniques: Flow Cytometry, Transformation Assay, Comparison

Journal: Communications Biology

Article Title: Vaccine-elicited IL-1R signaling results in Th17 TRM-mediated immunity

doi: 10.1038/s42003-024-06138-0

Figure Lengend Snippet: a CD4 T cells, b B cells, and c Th17 cells in 50,000 events from the lungs of vaccinated and unvaccinated mice either untreated (Naïve) or treated with isotype antibody, anti-IL-1α, or anti-IL-1β, as measured in flow cytometry ( n = 8 mice, Isotype; n = 6 mice, naïve; n = 9 mice, anti-IL-1α and anti-IL-1β). d Representative dot plots of Th17 cells (CD3 + , CD4 + , TCR-β + , IL-17A + ) in untreated (Naïve) and antibody- or isotype control-treated mice. e ELISpot measuring IL-17A secreting cells after overnight stimulation with OmpX. Data are displayed as spot counts (SFU) per 10 5 plated cells from single cell lung suspensions ( n = 3 mice, naïve; n = 4 mice for all other groups). f Serum IgG titers specific to OmpX from each vaccinated group ( n = 3 mice, naïve; n = 4 mice for all other groups). g Area under the curve of the data depicted in f . Log transformed bacterial burdens in the ( h ) lungs and ( i ) spleens 24 h post-challenge were determined using cfu plating assays ( n = 6 mice, naïve; n = 9 mice, all other groups). All data are presented as mean +/− SEM. Statistical differences were determined using a one-way ANOVA followed by Tukey’s multiple comparison test.

Article Snippet: Antibodies used for blocking and staining are as follows: Rat Anti-Mouse CD16/CD32 Fc Block (clone 2.4G2, BD Biosciences, Cat #553141), PE-Cy7 Rat Anti-Mouse CD4 (clone RM4-5, BD Biosciences, Cat #561099), APC rat anti-mouse CD3e (clone 17A2, BioLegend, Cat #100236), PE-Cy5 hamster anti-mouse TCRβ (clone H57-597, BD Biosciences, Cat #553173), FITC rat anti-mouse IL-17A (clone TC11-18H10.1, BioLegend, Cat #506907), Brilliant Violet 421 rat anti-mouse IFNγ (clone XMG1.2, BioLegend, Cat #505829), PE rat anti-mouse B220 (clone RA3-6B2, BioLegend, Cat #103207).

Techniques: Flow Cytometry, Enzyme-linked Immunospot, Transformation Assay, Comparison

Journal: eLife

Article Title: The kinase PDK1 is critical for promoting T follicular helper cell differentiation

doi: 10.7554/eLife.61406

Figure Lengend Snippet:

Article Snippet: Antibody , Rat monoclonal anti-mouse CD4-PE/Cy7 , Thermo Fisher Scientific , Cat # 25-0041-82; RRID: AB_469576 , FACS (1:100).

Techniques: Isolation, Transfection, Construct, Plasmid Preparation, Sequencing, Recombinant, Synthesized, Staining, SYBR Green Assay, Adjuvant, Software