rabbit anti nk1  (Alomone Labs)

Journal: British Journal of Pharmacology

Article Title: Substance P as a putative efferent transmitter mediates GABAergic inhibition in mouse taste buds) Substance P as a putative efferent transmitter mediates GABAergic inhibition in mouse taste buds

doi: 10.1111/bph.14142

Figure Lengend Snippet: SP stimulates its receptors on Type I cells and induces Ca 2+ release from intracellular stores. (A) Bath‐application of 10 nM SP elicits Ca 2+ mobilization in Fura 2‐loaded taste cells (↓, SP). RP67580 (0.1 μM), an NK1 receptor antagonist (present throughout the shaded area), reversibly inhibited SP‐induced Ca 2+ responses. (B) Summary of SP‐elicited Ca 2+ responses before, during and after the presence of RP67580, plotted as in (A). Points represent normalized peak taste cell responses. Blue symbols show mean ± 95% confidence interval (95% CI). * P

Article Snippet: We used rabbit anti‐NK1 (1:200; catalogue #ATR‐001; Alomone Labs, Jerusalem, Israel) for NK1 receptors (Muñoz et al., ), guinea pig anti‐GLAST (1:200; catalogue #AB‐2571717; Frontier Institute, Hokkaido, Japan) for Type I cells (Yoshida et al., ) and rabbit anti‐GABA (1:1500; catalogue #A2050; Sigma) for taste cell transmitter (Dvoryanchikov et al., ; Huang et al., ).

Techniques:

Journal: Cell reports

Article Title: Neurokinin-1 Receptor Signaling Is Required for Efficient Ca2+ Flux in T-Cell-Receptor-Activated T Cells

doi: 10.1016/j.celrep.2020.02.054

Figure Lengend Snippet: T Cells Express the f-NK1R that Is Recruited at the Immune Synapse (A) NK1R (extracellular) expression by FACS on WT T cells and control NK1R KO T cells. (B) FACS analysis of NK1R (C terminus) on permeabilized WT T cells, untreated or after 24-h activation with CD3 and CD28 Ab. (C) Western blot of NK1R in T cells untreated or after 24 h incubation with CD3 and CD28 Ab. The 75-kDa band corresponds to glycosylated f-NK1R. One representative experiment of 3. Bar diagram: relative density of f-NK1R normalized to GAPDH. Results pooled from two experiments. Means ± 1 SD. (D) ImageStream of doublets of OT-II CD4 T cells and B6 DC loaded with OVA323–339 or not (Control). SP, HK-1, and the NK1R concentrate at the T cell-DC synapse (light blue mask), identified by rearrangement of F-actin labeled with Texas red-phalloidin. (E) Comparison by ImageStream of relative fluorescence intensities of phalloidin, SP, HK-1, and NK1R within the interface mask on doublets of OT-II T cells and B6 DC loaded with OVA 323–339 (+ OVA) or not (Control). (F) ImageStream of doublets of OT-II T cells and B6 NK1R KO DC loaded or not (Control) with OVA 323–339 . The NK1R expressed by OT-II cells concentrates at the T cell-DC synapse (light blue mask). In (A) and (B), one representative experiment of 3. In (D)–(F), 1 of 2 experiments with 5,000 cells collected in each. Data were analyzed by 1-way ANOVA followed by ad hoc Student Newman Keuls test (C) and 2-tailed Student’s t test (E). *p

Article Snippet: Cells were surface labeled with APCCy7-CD11c Ab (1:100) and ATTO488-NK1R Ab against the extracellular domain of the NK1R (Alomone Labs,1:100), washed wi th ice-cold 5% FBS / PBS without EDTA, and fixed with 1.5% paraformaldehyde (30 min, RT).

Techniques: Expressing, FACS, Activation Assay, Western Blot, Incubation, Labeling, Fluorescence

Journal: Cell reports

Article Title: Neurokinin-1 Receptor Signaling Is Required for Efficient Ca2+ Flux in T-Cell-Receptor-Activated T Cells

doi: 10.1016/j.celrep.2020.02.054

Figure Lengend Snippet: Effect of NK1R-signaling on Polarization of CD4 T Cells (A) FACS analysis of proliferation (CFSE dilution) and intracellular cytokines in Th1 (IFN-γ)-, Th2 (IL-4)-, and Th17 (IL-17A)-polarized WT and NK1R KO CD4 T cells. (B) Comparison by FACS of expression of Th1 (T-bet), Th2 (GATA-3), and Th17 (RoRγt) transcription factors between WT and NK1R KO CD4 T cells cultured under polarizing conditions. (C) Assessment by FACS of cell death (by FVD incorporation) in WT and NK1R KO CD4 T cells polarized in vitro into Th1, Th2, or Th17 cells. In (A)–(C), numbers are cell percentages per quadrant. One representative experiment out of 3. (D) Quantification of WT and NK1R KO CD4 T cells expressing T-bet (Th1), GATA-3 (Th2), or RoRγt (Th17) after culture under polarizing conditions, analyzed by FACS. Each dot represents an independent experiment. Means ± 1 SD. (E) Quantification of cell death (by FVD incorporation) in proliferating (CFSE Low ) WT and NK1R KO CD4 T cells from (C). Each dot represents an individual experiment. Means ± 1 SD. (F) Concentrations of cytokines in supernatants of WT and NK1R KO CD4 T cells cultured for 4 days under polarizing conditions. Duplicates from 1 experiment representative of 3. Means ± 1 SD. (G) Secretion of IFN-γ and IL-17 by T cells homing in draining lymph nodes of skin sensitized with DNCB 5 days prior. Means ± 1 SD of 4 mice per variable. Data were analyzed by 1-way ANOVA followed by ad hoc Student Newman Keuls test (D–G). *p

Article Snippet: Cells were surface labeled with APCCy7-CD11c Ab (1:100) and ATTO488-NK1R Ab against the extracellular domain of the NK1R (Alomone Labs,1:100), washed wi th ice-cold 5% FBS / PBS without EDTA, and fixed with 1.5% paraformaldehyde (30 min, RT).

Techniques: FACS, Expressing, Cell Culture, In Vitro, Mouse Assay

Journal: Cell reports

Article Title: Neurokinin-1 Receptor Signaling Is Required for Efficient Ca2+ Flux in T-Cell-Receptor-Activated T Cells

doi: 10.1016/j.celrep.2020.02.054

Figure Lengend Snippet: Autocrine SP and HK-1 Promote IL-2 Secretion and Survival of T Cells (A) Quantification of Tac1 and Tac4 transcripts by RT-qPCR in CD4 or CD8 T cells before and following stimulation with CD3 and CD28 Ab during 2, 4, 6, and 24 h. Means ± 1 SD of 2 independent experiments. (B) ImageStream of cell doublets composed of OT-II CD4 T cells and Tac1 / 4 Double KO B6 BMDC loaded with OVA 323–339 . SP and HK-1 concentrate within the area of the T cell-DC synapse (light blue mask) identified by rearrangement of F-actin visualized by staining with Texas red-phalloidin. (C) Concentration of IL-2 (by ELISA) in 24-h supernatants of WT, NK1R KO , and Tac1 / 4 Double KO T cells cultured untreated or with CD3 and CD28 Ab. Means ± 1 SD of 3 experiments. (D) Concentrations of IL-2 (by ELISA) in supernatants of T cells cultured untreated, with CD3 and CD28 Ab alone or plus exogenous SP or HK-1. Means ± 1 SD of 3 experiments. Results were analyzed by 1-way ANOVA followed by ad hoc Student Newman Keuls test (A, C, and D). **p

Article Snippet: Cells were surface labeled with APCCy7-CD11c Ab (1:100) and ATTO488-NK1R Ab against the extracellular domain of the NK1R (Alomone Labs,1:100), washed wi th ice-cold 5% FBS / PBS without EDTA, and fixed with 1.5% paraformaldehyde (30 min, RT).

Techniques: Quantitative RT-PCR, Staining, Concentration Assay, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Cell reports

Article Title: Neurokinin-1 Receptor Signaling Is Required for Efficient Ca2+ Flux in T-Cell-Receptor-Activated T Cells

doi: 10.1016/j.celrep.2020.02.054

Figure Lengend Snippet: NK1R-signaling Promotes Activation of NFAT1, NFAT2, and NFκb-p65, and T Cell Survival Dependent on IL-2 Secretion (A) Calcineurin activity in WT and NK1R KO T cells incubated with SarSP or CD3 Ab, measured 15 min after treatment. Means ± 1 SD of duplicated results of 1 representative experiment of 2. (B) Western blot analysis of NFAT1, NFAT2, NFκβ-p65, cFos, and cJun, in nuclear extracts of WT and NK1R KO T cells, untreated or after incubation with CD3 and CD28 Ab. Controls were stimulated with ionomycin (Iono). One representative experiment of 3. (C) Concentrations of IL-2 by ELISA in supernatants of WT and NK1R KO T cells untreated or after 24-h stimulation with CD3 and CD28 Ab. Means ± 1 SD, 1 representative of 3 experiments. (D) Surface IL-2Rα expression by FACS in WT and NK1R KO T cells untreated or after 24-h stimulation with CD3 and CD28 Ab. One representative of 3 experiments. (E) Comparison by FACS of T cell proliferation (CFSE dilution), cell death (FVD incorporation), and activation (CD44 High ) between WT and NK1R KO T cells after 4-day stimulation with CD3 and CD28 Ab. Numbers are cell percentages per quadrant. One representative out of 6 experiments. (F) Comparison by FACS of cycles of cell division (histograms) and percentages of cell proliferation (bar diagram) between WT and NK1R KO T cells. Numbers in histograms are cell percentages. Each dot in the bar diagrams represents a mouse. Means ± 1 SD. (G) Percentages of cell death (by FVD incorporation) of WT and NK1R KO T cells stimulated for 4 days with CD3 and CD28 Ab, alone or plus IL-2. Means ± 1 SD, 6 mice per condition. Results were analyzed by 1-way ANOVA followed by ad hoc Student Newman Keuls test (A, C, F, and G). ***p

Article Snippet: Cells were surface labeled with APCCy7-CD11c Ab (1:100) and ATTO488-NK1R Ab against the extracellular domain of the NK1R (Alomone Labs,1:100), washed wi th ice-cold 5% FBS / PBS without EDTA, and fixed with 1.5% paraformaldehyde (30 min, RT).

Techniques: Activation Assay, Activity Assay, Incubation, Western Blot, Enzyme-linked Immunosorbent Assay, Expressing, FACS, Mouse Assay

Journal: Cell reports

Article Title: Neurokinin-1 Receptor Signaling Is Required for Efficient Ca2+ Flux in T-Cell-Receptor-Activated T Cells

doi: 10.1016/j.celrep.2020.02.054

Figure Lengend Snippet: NK1R-signaling Per Se Promotes Ca 2+ Flux in T Cells and Potentiates the Ca 2+ Flux Triggered by the T Cell Receptor (TCR) (A) Ca 2+ flux by ratiometric assay by FACS in T cells exposed to SarSP added after acquisition of the 30-s baseline (arrows). (B) Live-cell imaging of Ca 2+ flux in T cells exposed to SarSP alone or with the G αq/11 inhibitor YM-254,890. Ca 2+ flux was analyzed during 10 min after adding SarSP and images were acquired every 30 s. Representative cells out of 200. Heat bar: Fluo-4-AM fluorescence intensity from blue (minimum) to red (maximum) that correlates directly with Ca 2+ flux. Bar diagram: quantification of Fluo-4-AM signal on 200 individual cells, recorded for 10 min. Ca 2+ flux on each cell was calculated by the difference between the maximal and minimal intensity of Fluo-4-AM fluorescence (peak value). (C and D) Ratiometric assays of Ca 2+ flux by FACS in T cells from WT or global NK1R KO mice (C), or from WT or NK1R KO BM T cell chimeras (D) untreated of stimulated by CD3 Ab cross linking (arrows). Bar diagrams: area under the curve (AUC) of Ca 2+ flux. Each symbol corresponds to T cells from a different mouse analyzed in the same experiment. (E and F) Ratiometric assays of Ca 2+ flux by FACS in T cells untreated or exposed to the NK1R antagonists L733,060 (E) or WIN-51,708 (F) and stimulated by CD3 Ab cross linking (arrows). Bar diagrams: AUC of Ca 2+ flux. Each dot corresponds to T cells from a different mouse. (G) Ratiometric assay by FACS of intracellular Ca 2+ efflux in WT and NK1R KO T cells following stimulation by CD3 Ab cross linking (X, arrow) in Ca 2+ -free media. (H) Ratiometric assay of Ca 2+ influx by FACS in WT and NK1R KO T cells treated with thapsigargin (solid arrows) in Ca 2+ -free media for 4 min and then stimulated by CD3 Ab cross linking (X, dash arrows) in the presence of Ca 2+ . (G and H) One representative experiment out of 2. In (A) and (C)–(H), baselines were recorded for 30 s before addition of stimuli. Results were analyzed by 1-way ANOVA followed by ad hoc Student Newman Keuls test (C–H) and 2-tailed Student’s t test (B). **p

Article Snippet: Cells were surface labeled with APCCy7-CD11c Ab (1:100) and ATTO488-NK1R Ab against the extracellular domain of the NK1R (Alomone Labs,1:100), washed wi th ice-cold 5% FBS / PBS without EDTA, and fixed with 1.5% paraformaldehyde (30 min, RT).

Techniques: FACS, Live Cell Imaging, Fluorescence, Mouse Assay

Journal: Cell reports

Article Title: Neurokinin-1 Receptor Signaling Is Required for Efficient Ca2+ Flux in T-Cell-Receptor-Activated T Cells

doi: 10.1016/j.celrep.2020.02.054

Figure Lengend Snippet: In Vivo Signaling of the NK1R Sustains T Cell Survival during Priming and the Effector Phase of Contact Dermatitis (A) Proliferation (CFSE dilution), activation (CD44 High ), and cell death (FVD incorporation) of CFSE-labeled WT (CD45.2 Thy1.1) and NK1R KO (CD45.2 Thy1.2) T cells i.v. injected in B6 (CD45.1) mice, analyzed by FACS in inguinal lymph nodes draining skin sensitized with DNCB, 5 days prior. (B) Quantification of cell death (FVD incorporation) in i.v.-transferred CFSE-labeled WT and NK1R KO T cells that proliferated (CFSE Low ) in response to DNCB sensitization on the abdominal skin. Each dot represents inguinal lymph nodes pooled from an individual mouse. Means ± 1 SD, 4–5 mice per condition. Numbers are cell percentages per quadrant. (C) Images of tissue sections of the elicitation site (ear) of NK1R KO T cell and WT T cell BM chimeras, 48 h after DTH elicitation. The leukocyte infiltrate and edema (arrows) are more prominent in skin of control WT T cell BM chimeras. Insets: leukocyte infiltrate. H E, X 200–500. (D) Percentages of ear thickness increase in NK1R KO T cell and WT T cell BM chimeras after DTH elicitation. Means ± 1 SD, 10 mice per group. (E) Percentage of T cells in the CD45 gate of cell suspensions from the elicitation site (ear skin) of WT and NK1R KO T cell BM chimeras, analyzed 48 h after DTH elicitation. (F) Histological analysis of the DTH elicitation site (ear skin) of T cell BM chimeras showing T cells (green) undergoing apoptosis (TUNEL Pos , red). Nuclei were stained blue with DAPI. X 200–500. (G) Bar diagram with percentages of T cells labeled by TUNEL at the DTH elicitation site. Means ± 1 SD, 10 individual samples. (H) Images of tissue sections of the elicitation site (ear skin) of Tac1 / 4 Double KO T cell BM chimeras and control WT T cell BM chimeras collected 48 h after DTH elicitation. The elicitation site of control chimeras exhibited more prominent leukocyte infiltration and edema (arrows) than the skin of Tac1 / 4 Double KO T cell BM chimeras (H E, X 200). (I) Percentages of ear thickness increase in Tac1 / 4 Double KO T cell BM chimeras and control WT T cell BM chimeras, measured after 24, 48, 72, and 96 h following DTH elicitation. Means ± 1 SD of 10 mice per group. Data were analyzed by 1-way ANOVA followed by ad hoc Student Newman Keuls test (B, D, and I) and 2-tailed Student’s t test (E and G). *p

Article Snippet: Cells were surface labeled with APCCy7-CD11c Ab (1:100) and ATTO488-NK1R Ab against the extracellular domain of the NK1R (Alomone Labs,1:100), washed wi th ice-cold 5% FBS / PBS without EDTA, and fixed with 1.5% paraformaldehyde (30 min, RT).

Techniques: In Vivo, Activation Assay, Labeling, Injection, Mouse Assay, FACS, TUNEL Assay, Staining

Journal: Cell reports

Article Title: Neurokinin-1 Receptor Signaling Is Required for Efficient Ca2+ Flux in T-Cell-Receptor-Activated T Cells

doi: 10.1016/j.celrep.2020.02.054

Figure Lengend Snippet: NK1R-Signaling by Autocrine SP and HK-1 Promotes T Cell Survival (A) FACS analysis of proliferation (CFSE dilution) and activation (CD44 high ) of WT or Tac1 / 4 Double KO T cells, the latter unable to synthetize SP and HK-1, kept in culture for 4 days untreated or with CD3 and CD28 Ab. Numbers are cell percentages per quadrant. One representative experiment of 5. (B) Proliferation (CFSE dilution) and cell death (FVD incorporation) analyzed by FACS on WT and Tac1 / 4 Double KO T cells cultured for 4 days untreated or with CD3 and CD28 Ab alone, or plus exogenous SP, HK-1, or both. Numbers are cell percentages per quadrant. One representative of 3 experiments. (C) Quantification of cell death (FVD incorporation) in dividing (CFSE Low ) WT and Tac1 / 4 Double KO T cells from the experiments shown in (B). Each dot represents an independent experiment. Means ± 1 SD. Results in (C) were analyzed by 1-way ANOVA followed by ad hoc Student Newman Keuls test. *p

Article Snippet: Cells were surface labeled with APCCy7-CD11c Ab (1:100) and ATTO488-NK1R Ab against the extracellular domain of the NK1R (Alomone Labs,1:100), washed wi th ice-cold 5% FBS / PBS without EDTA, and fixed with 1.5% paraformaldehyde (30 min, RT).

Techniques: FACS, Activation Assay, Cell Culture