Journal: bioRxiv

Article Title: Paradox Found: Global Accounting of Lymphocyte Protein Synthesis

doi: 10.1101/2022.10.31.514539

Figure Lengend Snippet: (A) Primary human lymphocytes from three independent donors were cultured in PMA/ionomycin and IL2 (+PMA/I) or IL-2 only (-PMA/I) for up to 5 days. CD45 + cells were processed for flow RPM. (B) Primary human lymphocytes were cultured ex vivo as indicated, followed by a 15 minute treatment with vehicle, harringtonin (HAR, 5μg/mL), pactamycin (PA, 10μM), emetine (EME, 25μg/mL), or cycloheximide (CHX, 200μg/ml). Cells were harvested, and surface and RPM staining was performed. Gated on CD45 + cells. Error bars represent standard deviation of two independent experiments. (C) Radioactive amino acid incorporation (0.2 mCi/mL [ H]-Leu for 5 min) or RPM (as in B) in day 1 resting human lymphocytes. Error bars represent standard deviation of two independent experiments. (D) Radioactive amino acid incorporation and RPM in resting and activated human lymphocytes. RPM MFI values (gated on CD45 + cells) on the left, [ H]-Leu incorporation values (cpm) in the middle, and ratios of the activated to the resting cells on the right. Each point represents a single donor; bars indicate the mean from 3-5 independent donors.

Article Snippet: For mouse lymphocyte stains, antibodies were: CD3ε BV786 (clone 145-2C11, BD), CD4BV510 (clone RM4-5, BD), CD5 APC-R700 (clone 53-7.3, BD), CD8α PE-CF594 (clone 53-6.7, BD), CD11b PE-Cy7 (clone M1/70, eBioscience), CD19 APC-Cy7 (eBio1D3, eBioscience), CD25 BV650 (PC61, BD), CD44 BV605 (IM7, BD), CD44 eFl450 (clone IM7, eBioscience), CD45.1 APC (clone A20, eBioscience), CD45.1 eFl450 (clone A20, eBioscience), CD45.2 eFluor450 (clone 104, eBioscience), CD45.2 PE-Cy7 (clone 104, eBioscience), CD69 PerCP-Cy5.5 (clone H1.2F3, Invitrogen), γδ TCR PE (eBioGL3, GL3, eBioscience), Gr1 PE (clone RB6-8C5, BD), NK1.1 FITC (clone PK136, eBioscience), TCRβ 711 (H57-597, BD), and Vβ5.1/Vβ5.2 PE (clone MR9-4, BD).

Techniques: Cell Culture, Ex Vivo, Staining, Standard Deviation

Journal: bioRxiv

Article Title: Paradox Found: Global Accounting of Lymphocyte Protein Synthesis

doi: 10.1101/2022.10.31.514539

Figure Lengend Snippet: (A) Population frequency and RPM of resting D2 or D5 human lymphocytes or PMA/ionomcyin and IL2 activated D2 or D5 human lymphocytes. Left panel is the percent of CD45 + cells in the indicated population, the right panel is the amount of RPM signal in each population. (B) Representative RPM flow cytometry plot gated on CD8 + T cellsSimilar data was obtained from all donors.

Article Snippet: For mouse lymphocyte stains, antibodies were: CD3ε BV786 (clone 145-2C11, BD), CD4BV510 (clone RM4-5, BD), CD5 APC-R700 (clone 53-7.3, BD), CD8α PE-CF594 (clone 53-6.7, BD), CD11b PE-Cy7 (clone M1/70, eBioscience), CD19 APC-Cy7 (eBio1D3, eBioscience), CD25 BV650 (PC61, BD), CD44 BV605 (IM7, BD), CD44 eFl450 (clone IM7, eBioscience), CD45.1 APC (clone A20, eBioscience), CD45.1 eFl450 (clone A20, eBioscience), CD45.2 eFluor450 (clone 104, eBioscience), CD45.2 PE-Cy7 (clone 104, eBioscience), CD69 PerCP-Cy5.5 (clone H1.2F3, Invitrogen), γδ TCR PE (eBioGL3, GL3, eBioscience), Gr1 PE (clone RB6-8C5, BD), NK1.1 FITC (clone PK136, eBioscience), TCRβ 711 (H57-597, BD), and Vβ5.1/Vβ5.2 PE (clone MR9-4, BD).

Techniques: Flow Cytometry

Journal: bioRxiv

Article Title: Paradox Found: Global Accounting of Lymphocyte Protein Synthesis

doi: 10.1101/2022.10.31.514539

Figure Lengend Snippet: (A) Schematic representation of the ribopuromycylation (RPM) Ribosome Transit Analysis (RTA) method. Translation initiation is blocked and the decrease in RPM is monitored by flow cytometry as the elongating ribosomes run off mRNA. (B) RPM-RTA in HeLa cells. Harringtonin (HAR, 5μg/mL) is used to inhibit new ribosome initiation; emetine (EME, 25μg/mL) is used to freeze ribosomes on mRNA; puromycin (PMY, 50μg/mL) generates RPM signal. Curve is fitted using one phase exponential decay, and ribosome transit times are expressed as RPM half-time to decay. (C) Same as B, but cells are instead lysed in the presence of MG-132 and subjected to anti-puromycin western blot analysis. (D) Representative plots of the RPM-RTA signal in resting and activated human CD45+ lymphocytes (left three panels). Far right, ribosome transit times determined from 3 independent donors. Each dot represents data from one individual donor; the horizontal bars indicate the mean. (E) Ribosome transit times as in A but determined by [ H]-Leu incorporation instead of RPM. After treatment with HAR or HAR plus EME, cells were labeled for 5 minutes in 0.25mCi/mL [ H]-Leu. Right panel, ribosome transit times determined by [ H]-Leu incorporation from three independent donors. Each dot represents data from one individual donor; the horizontal bars indicate the mean.

Article Snippet: For mouse lymphocyte stains, antibodies were: CD3ε BV786 (clone 145-2C11, BD), CD4BV510 (clone RM4-5, BD), CD5 APC-R700 (clone 53-7.3, BD), CD8α PE-CF594 (clone 53-6.7, BD), CD11b PE-Cy7 (clone M1/70, eBioscience), CD19 APC-Cy7 (eBio1D3, eBioscience), CD25 BV650 (PC61, BD), CD44 BV605 (IM7, BD), CD44 eFl450 (clone IM7, eBioscience), CD45.1 APC (clone A20, eBioscience), CD45.1 eFl450 (clone A20, eBioscience), CD45.2 eFluor450 (clone 104, eBioscience), CD45.2 PE-Cy7 (clone 104, eBioscience), CD69 PerCP-Cy5.5 (clone H1.2F3, Invitrogen), γδ TCR PE (eBioGL3, GL3, eBioscience), Gr1 PE (clone RB6-8C5, BD), NK1.1 FITC (clone PK136, eBioscience), TCRβ 711 (H57-597, BD), and Vβ5.1/Vβ5.2 PE (clone MR9-4, BD).

Techniques: Flow Cytometry, Western Blot, Labeling

Journal: bioRxiv

Article Title: Paradox Found: Global Accounting of Lymphocyte Protein Synthesis

doi: 10.1101/2022.10.31.514539

Figure Lengend Snippet: (A) Depiction of the RPM-RTA in vivo method. Labeled OT-I T cells are first adoptively transferred, followed by VACV-SIINFEKL infection. RTA analysis is performed by intravenous injection of HAR and PMY (+/-CHX to prevent leakiness from HAR inhibition alone). Spleens are harvested for RPM analysis on both endogenous and transferred T cells. (B) CFSE-labeled Ly5.2 + (CD45.2 + CD45.1 - ) OT-I T cells were adoptively transferred into Ly5.1 (CD45.1 + CD45.2 - ) mice, which were then infected with VACV-SIINFEKL to activate the OT-I cells. Three days after infection, mice were intravenously injected with HAR simultaneously with PMY for 5 minutes (maximum signal), or first injected with HAR for ∼110, ∼275, or ∼575 seconds before being injected with PMY for 5 minutes. Splenocytes from mice were harvested, surface stained for gating and activation markers as indicated, fixed and permeabilized, and stained for RPM. Gates were CFSE lo OT-I CD8 + T cells to measure decay in activated cells, and CD44 - CD8 + or CD44 - CD4 + T cells to measure decay in resting T cells. The curve was generated by fitting to a one phase exponential decay. Representative of two independent experiments, 2-4 mice per group, with the mean and standard deviation of the calculated half-life decays as indicated. (C) RTA, with CHX modification, of adoptively transferred OT-I T cells or un-activated host CD8 + T cells in mice infected for 2 or 3 days with VACV-SIINFEKL. 3-4 independent experiments combined, normalized by setting maximum background-subtracted signal to 100.

Article Snippet: For mouse lymphocyte stains, antibodies were: CD3ε BV786 (clone 145-2C11, BD), CD4BV510 (clone RM4-5, BD), CD5 APC-R700 (clone 53-7.3, BD), CD8α PE-CF594 (clone 53-6.7, BD), CD11b PE-Cy7 (clone M1/70, eBioscience), CD19 APC-Cy7 (eBio1D3, eBioscience), CD25 BV650 (PC61, BD), CD44 BV605 (IM7, BD), CD44 eFl450 (clone IM7, eBioscience), CD45.1 APC (clone A20, eBioscience), CD45.1 eFl450 (clone A20, eBioscience), CD45.2 eFluor450 (clone 104, eBioscience), CD45.2 PE-Cy7 (clone 104, eBioscience), CD69 PerCP-Cy5.5 (clone H1.2F3, Invitrogen), γδ TCR PE (eBioGL3, GL3, eBioscience), Gr1 PE (clone RB6-8C5, BD), NK1.1 FITC (clone PK136, eBioscience), TCRβ 711 (H57-597, BD), and Vβ5.1/Vβ5.2 PE (clone MR9-4, BD).

Techniques: In Vivo, Labeling, Infection, Injection, Inhibition, Staining, Activation Assay, Generated, Standard Deviation, Modification

Journal: bioRxiv

Article Title: Paradox Found: Global Accounting of Lymphocyte Protein Synthesis

doi: 10.1101/2022.10.31.514539

Figure Lengend Snippet: (A) C57BL/6 mice were treated intravenously with CHX and PMY or only PMY. After the indicated times, splenocytes were harvested, surface stained, fixed/permeabilized, and RPM staining was performed. Representative of three independent experiments, 2-3 mice per group. (B) In one set of C57BL/6 mice, HAR was intravenously injected for 15 minutes before intravenously injecting mice with PMY for 5 min. In a second set of mice, CHX and PMY were IV injected for 5 min. Splenocytes from each set of mice were harvested, surface stained, fixed, and permeabilized, and RPM staining was performed for various immune cell subsets. To determine relative amounts of ribosomes, the ‘HAR then PMY’ RPM signal was subtracted from the CHX+PMY RPM signal for each cell subset after flow cytometry. Representative of two independent experiments, 2-4 mice per group. (C) CFSE-labeled Ly5.2 + (CD45.2 + CD45.1 - ) OT-I cells were adoptively transferred into Ly5.1 + (CD45.1 + CD45.2 - ) mice, which were then infected with VAC-SIINFEKL. 1-3 days after infection, mice were intravenously injected with CHX simultaneously with PMY for 5 min. Splenocytes from the mice were harvested, surface stained, fixed and permeabilized, and RPM staining was performed. Representative flow cytometry plots gated on OT-I T cells. (D) Number of divisions (by CFSE dilution) of OT-I T cells 1-3 days after infection of mice with VACV-SIINFEKL. (E) Amount of translation as measured by RPM signal (with no,PMY signal subtracted) in uninfected, or one-, two-, or three-day VACV-SIINFEKL-infected mice. Representative of four independent experiments, 2-3 mice per time point.

Article Snippet: For mouse lymphocyte stains, antibodies were: CD3ε BV786 (clone 145-2C11, BD), CD4BV510 (clone RM4-5, BD), CD5 APC-R700 (clone 53-7.3, BD), CD8α PE-CF594 (clone 53-6.7, BD), CD11b PE-Cy7 (clone M1/70, eBioscience), CD19 APC-Cy7 (eBio1D3, eBioscience), CD25 BV650 (PC61, BD), CD44 BV605 (IM7, BD), CD44 eFl450 (clone IM7, eBioscience), CD45.1 APC (clone A20, eBioscience), CD45.1 eFl450 (clone A20, eBioscience), CD45.2 eFluor450 (clone 104, eBioscience), CD45.2 PE-Cy7 (clone 104, eBioscience), CD69 PerCP-Cy5.5 (clone H1.2F3, Invitrogen), γδ TCR PE (eBioGL3, GL3, eBioscience), Gr1 PE (clone RB6-8C5, BD), NK1.1 FITC (clone PK136, eBioscience), TCRβ 711 (H57-597, BD), and Vβ5.1/Vβ5.2 PE (clone MR9-4, BD).

Techniques: Staining, Injection, Flow Cytometry, Labeling, Infection

Journal: Cell reports

Article Title: Chronic stress physically spares but functionally impairs innate-like invariant T cells

doi: 10.1016/j.celrep.2021.108979

Figure Lengend Snippet: (A) WT B6 mice were left undisturbed or restrained for 12 h, after which HMNCs and splenocytes were harvested and stained with a monoclonal antibody (mAb) to TCRβ along with empty (control) or PBS-57-loaded CD1d tetramers. Representative dot plots and summary data depict hepatic and splenic i NKT cell frequencies in stressed and control mice. (B) The absolute numbers of i NKT and TCRβ + PBS-57-loaded CD1d tetramer − T conv cells were also calculated. (C) In addition, the percentages of i NKT and T conv cells containing active caspases were determined by flow cytometry. (D) Hepatic i NKT and T conv cells were purified from ≥5 mice that had been either subjected to 2, 6, or 12 h of restraint stress or left undisturbed. After obtaining cDNA, the indicated gene products were amplified by quantitative PCR. Gene expression fold changes in i NKT and T conv cells isolated from stressed mice relative to corresponding cell populations from control animals were calculated using the 2 −(ΔΔCt) method and used to generate a heatmap. (E) Hepatic i NKT and T conv cells were analyzed for their intracellular Bcl-2 content. (F) Hepatic T conv cells were enumerated in Nr3c1 fl and Nr3c1 fl Lck cre mice that had been either restrained or left undisturbed. (G) Cohorts of WT B6 mice were given corticosterone (CS) or Veh in drinking water for 21 days before they were sacrificed for their livers and spleens, in which i NKT and T conv cells were enumerated. Each symbol in (A)–(C) and (E)–(G) represents an individual mouse, and error bars represent SEM. *p < 0.05, **p < 0.01, ****p < 0.0001 by unpaired Student’s t tests. NS, not significant.

Article Snippet: PE-Cy7-conjugated anti-mouse/rat Bcl-2 (Clone 10C4) , Thermo Fisher Scientific , Cat # 25-6992-42; RRID: AB_2573516.

Techniques: Staining, Flow Cytometry, Purification, Amplification, Real-time Polymerase Chain Reaction, Expressing, Isolation

Journal: Cell reports

Article Title: Chronic stress physically spares but functionally impairs innate-like invariant T cells

doi: 10.1016/j.celrep.2021.108979

Figure Lengend Snippet:

Article Snippet: PE-Cy7-conjugated anti-mouse/rat Bcl-2 (Clone 10C4) , Thermo Fisher Scientific , Cat # 25-6992-42; RRID: AB_2573516.

Techniques: Recombinant, Western Blot, Enzyme-linked Immunosorbent Assay, Staining, In Situ, Selection, Software

Journal: Immunity

Article Title: Heterogenous populations of tissue-resident CD8 + T cells are generated in response to infection and malignancy

doi: 10.1016/j.immuni.2020.04.007

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: PE/Cy7 anti-CD45.2 (104) , Thermo Fisher Scientific , Cat# 25-0454-82, RRID:AB_2573350.

Techniques: Virus, Expressing, Recombinant, Transfection, Infection, Software

Journal: Cell

Article Title: NKG2A blockade potentiates CD8 T-cell immunity induced by cancer vaccines

doi: 10.1016/j.cell.2018.10.028

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: CD45.2 – APC-Cy7 (104) , eBioscience , Cat#: 47–0454–82; RRID: AB_1272175.

Techniques: Blocking Assay, Purification, Virus, Recombinant, Staining, Transfection, Cell Stimulation, Adjuvant, Mutagenesis, Transgenic Assay, Marker, Sequencing, Expressing, Plasmid Preparation, Software, CRISPR

Journal: bioRxiv

Article Title: Loss of the intracellular enzyme QPCTL limits chemokine function and reshapes myeloid infiltration to augment tumor immunity

doi: 10.1101/2022.01.25.477769

Figure Lengend Snippet: a , b , c , Qpctl −/− and Qpctl +/+ littermate mice were inoculated with Qpctl −/− or Qpctl +/+ tumors, respectively. a , Schematic representation of the experimental protocol. b , EO771 growth was measured over time (n = 12 mice per group). c , LL/2 growth was measured over time (n = 7 mice per group). Gray lines represent individual mice. Overlay of fitting spline curves for each group is shown. Black dotted lines represent spline curves from Qpctl +/+ groups. d , e , LL/2 tumors were excised 14 days after tumor inoculation and analyzed by flow cytometry. d , Representative flow cytometry plots identifying Ly6C + monocytic cells (red circle) and F4/80 hi macrophages (blue circle) among tumor infiltrating leukocytes. e , Frequency of tumor infiltrating Ly6C + monocytes and number of Ly6C + monocytes, macrophages, neutrophils, T cells and NK cells is depicted, n = 15 or 13 ( Qpctl −/− ) mice per group. f , g,h, Qpctl −/− and Qpctl +/+ littermate mice were inoculated respectively with Qpctl −/− or Qpctl +/+ LL/2 tumors. CD45.1 + WT monocytes were transferred into tumor bearing mice, 12 days post tumor inoculation. Tumors were analyzed 2 days after monocyte transfer. f , Schematic representation of the experimental protocol. g , Representative flow cytometry plots identifying transferred monocytes (red circle) among leukocytes. h , Quantification of transferred monocytes is depicted (n = 10 mice per group). i , CCL7 PTMs were quantified in the plasma of WT mice treated with ctrl vehicle or QPCTL inhibitor (QPCTLi) for 4 days (n = 10 or 8 (QPCTLi) mice per group). j , WT mice received ctrl or QPCTLi for 4 days before LL/2 or EO771 tumor inoculation (preventative) or at day 7 after LL/2 inoculation (therapeutic). On day 14 after tumor cell inoculation, tumors were excised and the number of tumor-infiltrating Ly6C + monocytic cells determined by flow cytometry (n = 8 (LL/2 preventative); n = 8 or 9 (EO771 QPCTLi), n = 10 or 6 (LL/2 therapeutic QPCTLi) mice per group). k,l , WT mice received ctrl or QPCTLi starting 1 day before LL/2 or EO771 tumor inoculation. Tumor growth was measured overtime. n = 15 (LL/2 ctrl); n = 14 (LL/2 QPCTLi) n= 14 (EO771 Ctrl), n = 12 (EO771 QPCTLi). Gray lines represent individual mice. Overlay of fitting spline curves for each group is shown. Black dotted lines represent spline curves from the ctrl groups. Bars are medians and symbols individual mice. Data shown is representative of one experiment ( c , i , j,k,l ) or pooled from 2 experiments ( b , e,h ). All experiments were repeated independently (⩾2 times). ns, not significant, * p ⩽0.05, ** p ⩽0.01, *** p ⩽0.001. P values are from nonparametric Mann-Whitney test ( e,h,i ) or Two-way ANOVA test ( b , c,k,l ).

Article Snippet: Antibodies for flow cytometry of mouse samples were: anti-CD3e (clone 145-2C11 in PECF594 from BD Biosciences Cat# 562286 or in FITC, from eBiosciences Cat# 11-0031-82, or clone 500A2 in AF700, from BD Biosciences Cat# 557984); anti-B220/CD45R (clone RA3-6B2 in APCeF780 from eBiosciences Cat# 47-0452-82, in AF700 from BD Biosciences Cat# 557957 or in FITC, from BioLegend Cat# 103206); anti-CD45 (clone 30-F11 in PECy7 from eBiosciences Cat# 25-0451-82 or in BV786, from BD Biosciences Cat# 564225); anti-CD45.1 (clone A20 in APC from BioLegend Cat# 110714); anti-CD45.2 (clone 104 in AF700 from eBiosciences Cat# 56-0454-82 or in APC from BioLegend Cat# 109814 or in pE-Cy7 from BioLegend Cat# 109830); anti-NK1.1 (clone PK136 in APCeF780 Cat# 47-5941-82 or APC cat# 17-5941-82 from eBiosciences, in AF700 from BD Biosciences Cat# 560515 or in FITC from BioLegend Cat# 108706); anti-CD11b (clone M1/70 in BV421, BD Biosciences Cat# 562605); anti-CD115/CSF1R (clone AFS98 in PECy7 from BioLegend Cat# 135524); anti-CCR2 (clone 475301 in APC from R&D systems Cat# FAB5538A); anti-CX3CR1 (clone SA011F11 in BV786 from Biolegend Cat# 149029); anti-F4/80 (clone T45-2342 in BUV395 from BD Biosciences Cat# 565614); anti-Ly6C (in PerCPCy5.5, clone HK1.4 from BioLegend Cat# 128012 or clone AL-21 from BD Biosciences Cat# 560525); anti-Ly6G (clone 1A8 in AF700, from BioLegend Cat# 127622); anti-MHC class II (I-A/I-E) (clone M5/114.15.2 in APCeF780 from eBiosciences Cat# 47-5321-82); anti-Siglec F (clone E50-2440 in PeCF594 from BD Biosciences Cat# 562757 or clone 1RNM44N in AF700 from Invitrogen Cat# 56-1702-82); anti-CD11c (clone N4.11 in PE, from eBiosciences Cat# 12-0114-82); Anti-Ki67 (clone SolA15 in PECy7 from Invitrogen Cat# 25-5698-82) and anti-Granzyme B (clone GB12 in APC, from Invitrogen Cat# MHGB05).

Techniques: Flow Cytometry, MANN-WHITNEY

Journal: eLife

Article Title: Neutrophils promote CXCR3-dependent itch in the development of atopic dermatitis

doi: 10.7554/eLife.48448

Figure Lengend Snippet: ( A ) Exact permutation test (10,000 iterations, see Materials and methods) for significance of mean absolute log 2 fold change in gene expression at Day 8 (MC903 vs. ethanol) of custom-defined groups of genes for indicated categories (see ). ( B ) Log 2 fold change in gene expression (MC903 vs. ethanol) in mouse skin at indicated time points for key immune and mouse/human AD genes that were significantly differentially expressed for at least one time point in the MC903 model. Only genes from our initial list (see Materials and methods) differentially expressed at corrected p<0.05 and changing >2 fold between treatments for at least one condition are shown. Green bars = increased expression in MC903 relative to ethanol; magenta = decreased expression. Exact values and corrected p -values are reported in and Supplemental Data, respectively. D1 = 6 hr post-treatment; D2 = Day 2; D5 = Day 5; D8 = Day 8. ( C ) Scratching behavior of mice treated with MC903 or ethanol for indicated length of time (two-way ANOVA: **** p interaction <0.0001, F(2,409) = 13.25; Sidak’s multiple comparisons: p day 3 = 0.1309, n = 62,51 mice; * p day 5 = 0.0171, n = 69,56 mice; **** p day 8 < 0.0001, n = 92,85 mice). Exact values displayed in . ( D ) Log 2 fold change in gene expression of neutrophil chemoattractants (upper), Th2 cytokines (middle) and T cell chemoattractants (lower, from RNA-seq data). ( E ) Neutrophil counts in MC903- and ethanol-treated skin at indicated time points (two-way ANOVA: ** p treatment = 0.0023, F(1,102) = 9.82; Sidak’s multiple comparisons: p day 2 > 0.999, n = 4,4 mice; p day 3 = 0.9801, n = 5,5 mice; *** p day 5 = 0.0003, n = 6,8 mice; *** p day 8 = 0.0001, n = 40,38 mice). ( F ) Basophil counts in MC903- and ethanol-treated skin at indicated time points (two-way ANOVA: ** p treatment = 0.0051, F(1,102) = 8.17; Sidak’s multiple comparisons: p day 2 > 0.999, n = 4,4 mice; p day 3 = 0.8850, n = 5,5 mice; p day 5 = 0.0606, n = 6,8 mice; **** p day 8 < 0.0001, n = 40,38 mice). ( G ) CD4 + T cell counts in MC903- and ethanol-treated skin at indicated time points (two-way ANOVA: ** p time = 0.0042, F(1,44) = 9.10; p day 3 = 0.9998, n = 8,6 mice; p day 5 = 0.2223, n = 9,8 mice; ** p day 8 = 0.0021, n = 11,8 mice). Day 8 immune cell infiltrate represented as % of CD45 + cells in (see for all experimental conditions). Exact values displayed in and representative FACS plots for myeloid and T cell gating shown in and . For , data from mice receiving i.p. injection of PBS (see ) in addition to MC903 or EtOH are also included. ( H ) (Upper and Lower) Representative maximum intensity Z-projections from immunohistochemistry (IHC) of whole-mount mouse skin on Day 2 of the MC903 model. Skin was stained with neuronal marker beta-tubulin III (BTIII; green). Hair follicle autofluorescence is visible in the magenta channel. Images were acquired on a confocal using a 20x water objective. ( I ) Quantification of innervation (see Materials and methods) of mouse skin as determined from BTIII staining (*p=0.012; two-tailed t-test ( t = 3.114; df = 9); n = 7,4 images each from two mice per treatment). Day 1 IHC results as follows: 31.78 ± 18.39% (MC903) and 31.51 ± 16.43% (EtOH); p=0.988; two-tailed unpaired t-test; n = 6 images each from two mice per treatment. Exact values are reported in . ( J ) Quantification of CGRP + nerve fibers (see Materials and methods) in skin (**p=0.0083; two-tailed t-test ( t = 2.868; df = 25); n = 15, 12 images from three mice per treatment). Exact values are reported in . Representative images in . Figure 1—source data 1. Values displayed in the bar plot shown in . Figure 1—source data 2. Values displayed in the heat map shown in . Figure 1—source data 3. Values displayed in the bar plot shown in . Figure 1—source data 4. Values displayed in the bar plots shown in and . Figure 1—source data 5. Values displayed in the bar plots shown in and . Figure 1—source data 6. Values displayed in the heat map shown in . Figure 1—source data 7. Values displayed in the heat map shown in . Figure 1—source data 8. Values displayed in the heat map shown in . Figure 1—source data 9. Values displayed in the bar plot shown in .

Article Snippet: Antibody , CD45.2 Monoclonal Antibody (104), APC-Cy7, eBioscience; CD45.2-APC/Cy7 (104) (Mouse monoclonal, 1:200) , eBioscience , RRID: AB_1272175 ; Cat # 47-0454-82 , .

Techniques: Expressing, RNA Sequencing Assay, Injection, Immunohistochemistry, Staining, Marker, Two Tailed Test

Journal: eLife

Article Title: Neutrophils promote CXCR3-dependent itch in the development of atopic dermatitis

doi: 10.7554/eLife.48448

Figure Lengend Snippet: ( A ) Representative flow cytometry plots of cells collected from blood of mice injected with PBS or aGr1 (250 μg, i.p.) once-daily for five days concurrent with daily MC903 topical treatment. Shown are CD45.2 + CD11b + cells, plotted by Ly6G and Ly6C signal, with neutrophil (Neuts.) and inflammatory monocyte (IMs) populations indicated. Neutrophils were defined as Cd11b + Ly6G + Ly6C mid/high and IMs were defined as Cd11b + Ly6G - Ly6C high (see Materials and methods). ( B ) Representative flow cytometry plot as in A, depicting neutrophil and IM populations from blood collected on day 8. ( C ) (Left) Neutrophil counts in blood shown as % of Cd11b + cells from aGr1/MC903 (black triangles) and PBS/MC903 (gray circles)-treated animals on days 3, 5, and 8 of the model (two-way repeated measures ANOVA: **** p treatment <0.0001, F(1,31) = 299.5; Sidak’s multiple comparisons: **** p day 3 < 0.0001; **** p day 5 < 0.0001; **** p day 8 < 0.0001, n = 16,17 mice). (Right) Inflammatory monocyte counts in blood shown as % of Cd11b + cells from aGr1/MC903 and PBS/MC903-treated animals on days 3, 5, and 8 of the model (two-way repeated measures ANOVA: * p treatment = 0.0468, F(1,31) = 4.287; Sidak’s multiple comparisons: ** p day 3 = 0.0015; p day 5 = 0.1918; p day 8 = 0.2013, n = 16,17 mice). Exact values displayed in .

Article Snippet: Antibody , CD45.2 Monoclonal Antibody (104), APC-Cy7, eBioscience; CD45.2-APC/Cy7 (104) (Mouse monoclonal, 1:200) , eBioscience , RRID: AB_1272175 ; Cat # 47-0454-82 , .

Techniques: Flow Cytometry, Injection

Journal: eLife

Article Title: Neutrophils promote CXCR3-dependent itch in the development of atopic dermatitis

doi: 10.7554/eLife.48448

Figure Lengend Snippet: ( A ) Representative flow cytometry plots of cells from cheek skin of mice injected with PBS or CXCL1 (1 μg in 20 µL, s.c.). Shown are CD45.2 + CD11b + cells, plotted by Ly6G and Ly6C signal, with neutrophil and inflammatory monocyte (IMs) populations indicated. ( B ) Neutrophil count from cheek skin of mice 5, 15, and 30 min after injection of CXCL1 or PBS (two-way ANOVA: * p interaction = 0.0239, F(2,21) = 4.48; Sidak’s multiple comparisons: p 5 min >0.9999, n = 4,5 mice; * p day 15 min = 0.0141, n = 4,4 mice; ** p day 30 min = 0.0031, n = 3,7 mice). Exact values displayed in . ( C ) Blood neutrophils as % of Cd11b + cells approximately 20 hr after injection of 250 µg aGr1 (n = 15 mice). Mice assayed for CXCL1-evoked itch behavior immediately preceding blood isolation (see ). Exact values displayed in . See for representative blood neutrophil measurements from PBS-injected animals.

Article Snippet: Antibody , CD45.2 Monoclonal Antibody (104), APC-Cy7, eBioscience; CD45.2-APC/Cy7 (104) (Mouse monoclonal, 1:200) , eBioscience , RRID: AB_1272175 ; Cat # 47-0454-82 , .

Techniques: Flow Cytometry, Injection, Isolation

Journal: eLife

Article Title: Neutrophils promote CXCR3-dependent itch in the development of atopic dermatitis

doi: 10.7554/eLife.48448

Figure Lengend Snippet: ( A ) Exact permutation test (10,000 iterations, see Materials and methods) for significance of mean absolute log 2 fold change in gene expression at Day 8 (MC903 vs. ethanol) of custom-defined groups of genes for indicated categories (see ). ( B ) Log 2 fold change in gene expression (MC903 vs. ethanol) in mouse trigeminal ganglia (TG) at indicated time points for all genes which were significantly differentially expressed for at least one time point in the MC903 model. Green bars = increased expression in MC903 relative to ethanol; magenta = decreased expression. Exact values and corrected p -values are reported in and Supplemental Data, respectively. ( C ) Representative composite images showing immune cells (CD45, green), and sensory neurons (Prph, magenta) with DAPI (blue) in sectioned trigeminal ganglia from mice treated with Vehicle or MC903 for five days on the cheek. ( D ) Quantification of images examining average number of CD45 + cells per section and average ratio of CD45:Peripherin cells per section after five days of treatment (p=0.562 ( t = 0.6318, df = 4), 0.542 ( t = 0.6660, df = 4); two-tailed unpaired t-tests, n = 33–159 fields of view (images) each of both trigeminal ganglia from three mice per condition treated bilaterally). ( E ) Representative composite images showing immune cells (CD45, green), and sensory neurons (Peripherin (Prph), magenta) with DAPI (blue) in sectioned trigeminal ganglia from mice treated with Vehicle or MC903 for eight days on the cheek. ( F ) Quantification of images examining average number of CD45 + cells per section and average ratio of CD45:Peripherin cells per section after eight days of treatment (**p=0.0019 ( t = 5.977, df = 5), **p = 0.0093 ( t = 4.107, df = 4); two-tailed unpaired t-tests; n = 42–172 fields of view (images) each of both trigeminal ganglia from 3 EtOH or 4 MC903 animals treated bilaterally). Scale bar = 100 µm. Images were acquired on a fluorescence microscope using a 10x air objective. Values from bar plots and all TG IHC data are available in . ( G ) Log 2 fold change in gene expression (MC903 vs. ethanol) in mouse spinal cord on day 8 showing selected differentially expressed genes ( p adjusted < 0.05). Exact values and corrected p -values are reported in Supplemental Data. Figure 3—source data 1. Values displayed in the bar plot shown in . Figure 3—source data 2. Values displayed in the heat map shown in . Figure 3—source data 3. Quantification of all IHC samples from trigeminal ganglia, and Values displayed in the bar plots shown in .

Article Snippet: Antibody , CD45.2 Monoclonal Antibody (104), APC-Cy7, eBioscience; CD45.2-APC/Cy7 (104) (Mouse monoclonal, 1:200) , eBioscience , RRID: AB_1272175 ; Cat # 47-0454-82 , .

Techniques: Expressing, Two Tailed Test, Fluorescence, Microscopy

Journal: eLife

Article Title: Neutrophils promote CXCR3-dependent itch in the development of atopic dermatitis

doi: 10.7554/eLife.48448

Figure Lengend Snippet: ( A ) Representative composite image showing CD45 (green), Peripherin (magenta), and DAPI (blue). ( B ) Single-channel CD45 image with automated min/max intensity thresholding. ( C ) Resultant binary image generated from ( B ). ( D ) Cells were counted as the number of regions of interest (ROIs) outlined in blue.

Article Snippet: Antibody , CD45.2 Monoclonal Antibody (104), APC-Cy7, eBioscience; CD45.2-APC/Cy7 (104) (Mouse monoclonal, 1:200) , eBioscience , RRID: AB_1272175 ; Cat # 47-0454-82 , .

Techniques: Generated

Journal: eLife

Article Title: Neutrophils promote CXCR3-dependent itch in the development of atopic dermatitis

doi: 10.7554/eLife.48448

Figure Lengend Snippet:

Article Snippet: Antibody , CD45.2 Monoclonal Antibody (104), APC-Cy7, eBioscience; CD45.2-APC/Cy7 (104) (Mouse monoclonal, 1:200) , eBioscience , RRID: AB_1272175 ; Cat # 47-0454-82 , .

Techniques: Purification, Recombinant, Staining, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay, Titration, Protease Inhibitor, Flow Cytometry, Software

Journal: Immunity

Article Title: Megakaryocyte production is sustained by direct differentiation from erythromyeloid progenitors in the yolk sac until midgestation

doi: 10.1016/j.immuni.2021.04.026

Figure Lengend Snippet:

Article Snippet: CD45.2 APC-Cy7 (Clone 104) , SONY Biotechnology , Cat# 1149120; RRID: AB_830788.

Techniques: Blocking Assay, Recombinant, Sequencing, Software