unconjugated rabbit anti rat primary antibody  (R&D Systems)

Journal: iScience

Article Title: Mycobacterium tuberculosis impedes CD40-dependent notch signaling to restrict Th 17 polarization during infection

doi: 10.1016/j.isci.2022.104305

Figure Lengend Snippet: Engaging CD40 on Mtb-infected DCs augments pro-inflammatory cytokines and enhances expression of Notch ligand Dll4 BMDCs were infected with either media (UI) or Mtb H37Rv strain at an MOI of 1 in the presence or absence of 1 μg/mL of multimeric CD40L reagent (CD40LT). At designated time points, cellfree supernatants and RNA were collected to assay for cytokine secretion and mRNA transcript. (A) ELISA measurements of IL-6 and IL-12p40 in supernatants. (B and C) qPCR analysis of genes was standardized to the housekeeping gene GAPDH, analyzed using the ΔΔCt method, and presented as 2 -ΔΔCt . Data are presented as mean ± SD (a) or mean ± SEM (B and C). Data are representative of 3 independent experiments. Data were analyzed in (A) using a one-way ANOVA with a correction for multiple comparisons and (B and C) using a two-way ANOVA with a correction for multiple comparisons. Statistical significance p value key is the following: ∗ = ≤ 0.05, ∗∗∗∗ = ≤ 0.0001. See also Figures S1–S4 .

Article Snippet: BUV737 Rat Anti-Mouse DLL4 (Clone: 9A1.5) , BD , Catalog #: 748394; RRID: AB_2872813.

Techniques: Infection, Expressing, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: Mycobacterium tuberculosis impedes CD40-dependent notch signaling to restrict Th 17 polarization during infection

doi: 10.1016/j.isci.2022.104305

Figure Lengend Snippet: Engaging CD40 on Mtb-stimulated DCs enhances surface expression of DLL4 and Jagged1 BMDCs were stimulated with either media (UI) or HK Mtb (MOI 30) with or without the addition of 1 μg/mL of CD40LT. At designated time points, cells were collected and stained for surface markers. (A) Representative flow cytometry plot of DLL4 + and Jagged1 + frequencies. (B) Frequency of DLL4 + and Jagged1 + and double-positive populations. (C) MFI of DLL4 and Jagged1 expression and representative graphs. All populations are singlets/live cells/CD11c + MHCII hi . UI presented is 0H UI. Data are presented as mean ± SD. Data are representative of 3 independent experiments. Data were analyzed in (B) using a two-way ANOVA with a correction for multiple comparisons and (C) using an unpaired Student’s t test. Statistical significance p value key is the following: ∗ = ≤ 0.05, ∗∗ = ≤ 0.01, ∗∗∗ = ≤ 0.001, ∗∗∗∗ = ≤ 0.0001.

Article Snippet: BUV737 Rat Anti-Mouse DLL4 (Clone: 9A1.5) , BD , Catalog #: 748394; RRID: AB_2872813.

Techniques: Expressing, Staining, Flow Cytometry

Journal: iScience

Article Title: Mycobacterium tuberculosis impedes CD40-dependent notch signaling to restrict Th 17 polarization during infection

doi: 10.1016/j.isci.2022.104305

Figure Lengend Snippet: DLL4 is required for Th 17 polarization but is dispensable for Th 1 polarization BMDCs were stimulated with either media alone (UI), or HK Mtb (MOI 30) with or without 1 μg/mL of CD40LT. Following 24H of stimulation, cells were pulsed with 10 μg/mL of cognate peptide (OVA 323-339 ) for one hour and then co-cultured with purified naïve CD4 OT-II Tg Thy1.1 T cells at a ratio of 4:1 T cells to DCs. For blockade conditions, blocking antibodies to either DLL4, Jagged1, or both DLL4 and Jagged1 combined, were added at the time of co-culture at the following concentrations: 15, 30, or 60 μg/mL. After 72H of co-culture, cell-free supernatants were harvested and assayed for cytokines by ELISA. (A) IFN-γ (Th 1 ). (B) IL-17 (Th 17 ). (C) IL-2. Data are presented as mean ± SD. Data are representative of 3 independent experiments. Data were analyzed using a one-way ANOVA with a correction for multiple comparisons (all data points were compared to Mtb + CD40LT). Statistical significance p value key is the following: ∗∗∗ = ≤ 0.001, ∗∗∗∗ = ≤ 0.0001. See also Figures S5–S7 .

Article Snippet: BUV737 Rat Anti-Mouse DLL4 (Clone: 9A1.5) , BD , Catalog #: 748394; RRID: AB_2872813.

Techniques: Cell Culture, Purification, Blocking Assay, Co-Culture Assay, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: Mycobacterium tuberculosis impedes CD40-dependent notch signaling to restrict Th 17 polarization during infection

doi: 10.1016/j.isci.2022.104305

Figure Lengend Snippet: Blocking DLL4 singly or in combination with Jagged1 on DCs reduces antigen-specific IL-17 + CD4 T cell frequencies in vivo (A) Experimental schema. BMDCs we stimulated with either media alone (UI), or HK Mtb (MOI 30) with or without 1 μg/mL of CD40LT for 24H. At this time in the CD40LT condition, either DLL4 or DLL4 and Jagged1 blocking antibodies (30 μg/mL) were added. One day before transfer, 1E6 ESAT-6 transgenic T cells were transferred into mice via the intravenous (IV) route. On the day of intratracheal (IT) transfer, 1E6 DCs were transferred. At 6 days after transfer, mice were euthanized and lung single cell suspensions were stimulated with 10 μg/mL ESAT-6 1-20 peptide to assess antigen-specific responses. Cells were then stained for flow cytometry. (B) Representative flow cytometry plots of IFN-γ + and IL-17 + frequencies. (C) Cytokine-positive frequency of CD4 T cells. Populations shown are singlets/live cells/CD3 + /CD4 + . Experimental schema was made with BioRender.com . Data are presented as mean ± SD. Data are representative of 2 independent experiments. Data were analyzed using a one-way ANOVA with a correction for multiple comparisons. Statistical significance p value key is the following: ns = no significance, ∗ = ≤ 0.05, ∗∗ = ≤ 0.01, ∗∗∗ = ≤ 0.001, ∗∗∗∗ = ≤ 0.0001.

Article Snippet: BUV737 Rat Anti-Mouse DLL4 (Clone: 9A1.5) , BD , Catalog #: 748394; RRID: AB_2872813.

Techniques: Blocking Assay, In Vivo, Transgenic Assay, Staining, Flow Cytometry

Journal: iScience

Article Title: Mycobacterium tuberculosis impedes CD40-dependent notch signaling to restrict Th 17 polarization during infection

doi: 10.1016/j.isci.2022.104305

Figure Lengend Snippet: Blocking DLL4 on Mtb-infected DCs reduces Th 17 and multifunctional CD4 T cell responses in vivo (A) Experimental schema. BMDCs were infected with Mtb at an MOI of 1, with or without CD40LT, for 48H. For antibody blockade studies, 60 μg/mL anti-DLL4 antibody was added during infection. DCs were then harvested and 1E6 were intratracheally (IT) transferred into the lungs of mice along with additional blocking antibody. At 4 weeks post transfer, mice were euthanized and lung suspensions were unstimulated to asses ex vivo responses using flow cytometry. (B) Frequency of cytokine-positive CD4 T cells. (C) Boolean analysis of frequency of multiple cytokine-positive CD4 T cells. (D) Frequency of CXCR3 + CCR6 + CD4 T cells and Boolean analysis of CXCR3 + CCR6 + and cytokine-positive CD4 T cells. All populations are singlets/live cells/CD3 + /CD4 + . Experimental schema was made with BioRender.com . Data are presented as mean ± SD. Data are representative of 2 independent experiments. Data were analyzed using a one-way ANOVA with a correction for multiple comparisons. Statistical significance p value key is the following: ∗ = ≤ 0.05, ∗∗ = ≤ 0.01, ∗∗∗ = ≤ 0.001. SP = single-positive, DP = double-positive, TP = triple-positive. See also Figures S8 and .

Article Snippet: BUV737 Rat Anti-Mouse DLL4 (Clone: 9A1.5) , BD , Catalog #: 748394; RRID: AB_2872813.

Techniques: Blocking Assay, Infection, In Vivo, Ex Vivo, Flow Cytometry

Journal: iScience

Article Title: Mycobacterium tuberculosis impedes CD40-dependent notch signaling to restrict Th 17 polarization during infection

doi: 10.1016/j.isci.2022.104305

Figure Lengend Snippet: Engaging CD40 on Mtb-infected DCs increases NOTCH2 expression on CD4 T cells in the lung BMDCs were infected with Mtb at an MOI of 1, with or without CD40LT, for 48H. For antibody blockade studies, 60 μg/mL anti-DLL4 antibody was added during infection. DCs were then harvested and 1E6 were intratracheally (IT) transferred into the lungs of mice along with additional blocking antibody. At 4 weeks post transfer, mice were euthanized and lung suspensions were unstimulated to asses ex vivo responses using flow cytometry. (A) Frequency of NOTCH receptor-positive CD4 T cells. (B) Boolean analysis of NOTCH receptor-expressing and cytokine-positive CD4 T cells. All populations are singlets/live cells/CD3 + /CD4 + . Data are presented as mean ± SD. Data are representative of 2 independent experiments. Data were analyzed using a one-way ANOVA with a correction for multiple comparisons. Statistical significance p value key is the following: ∗ = ≤ 0.05, ∗∗ = ≤ 0.01, ∗∗∗ = ≤ 0.001, ∗∗∗∗ = ≤ 0.0001.

Article Snippet: BUV737 Rat Anti-Mouse DLL4 (Clone: 9A1.5) , BD , Catalog #: 748394; RRID: AB_2872813.

Techniques: Infection, Expressing, Blocking Assay, Ex Vivo, Flow Cytometry

Journal: iScience

Article Title: Mycobacterium tuberculosis impedes CD40-dependent notch signaling to restrict Th 17 polarization during infection

doi: 10.1016/j.isci.2022.104305

Figure Lengend Snippet: Th 17 responses correlate with NOTCH2 expression and lower lung CFU BMDCs were infected with Mtb at an MOI of 1, with or without CD40LT, for 48H. For antibody blockade studies, 60 μg/mL anti-DLL4 antibody was added during infection. DCs were then harvested and 1E6 were intratracheally (IT) transferred into the lungs of mice along with additional blocking antibody. At 4 weeks post transfer, mice were euthanized and lung suspensions were unstimulated to asses ex vivo responses using flow cytometry and lung homogenates were plated to enumerate Mtb burdens. (A) Correlogram using R package “corrplot” showing correlation between the frequency of marker-positive CD4 T cells and frequency of cytokine-positive CD4 T cells. (B) Correlations in R using “ggscatter” between the frequency of marker-positive CD4 T cells and frequency of cytokine-positive CD4 T cells. (C) Mtb lung colony-forming unit (CFU) and correlations between CFU and frequencies of marker-positive or cytokine-positive CD4 T cells using “ggscatter” in R. All correlations presented are Pearson’s correlations. Data in C) are presented as mean ± SD and were analyzed using an unpaired Student’s t test. Data are representative of 2 independent experiments. Statistical significance p value key is the following: ∗ = ≤ 0.05, ∗∗ = ≤ 0.01, ∗∗∗ = ≤ 0.001, ∗∗∗∗ = ≤ 0.0001. See also Figure S10 .

Article Snippet: BUV737 Rat Anti-Mouse DLL4 (Clone: 9A1.5) , BD , Catalog #: 748394; RRID: AB_2872813.

Techniques: Expressing, Infection, Blocking Assay, Ex Vivo, Flow Cytometry, Marker

Journal: iScience

Article Title: Mycobacterium tuberculosis impedes CD40-dependent notch signaling to restrict Th 17 polarization during infection

doi: 10.1016/j.isci.2022.104305

Figure Lengend Snippet: Mtb restricts DLL4 expression and early CD4 T cells responses in the lung through the Hip1 serine protease (A) Experimental schema. C57BL/6 mice were infected via the aerosol route with a low-dose of Mtb or hip1 mutant. Following 2 weeks post-infection, mice were euthanized and lung ex vivo responses were measured using flow cytometry. (B–D) (B) Frequency of CD40-expressing CD11b + DCs and CD40-expressing CD103 + DCs. Representative flow plots and frequency of DLL4 + , Jagged1 + , and DLL4 + Jagged1 + for (C) CD11b + DCs and (D) CD103 + DCs. (E) Frequency of cytokine-positive and NOTCH receptor-positive CD4 T cells. (F) Correlations between different CD4 T cell and innate immune population markers using “ggscatter” in R. All CD11b + DC populations are singlets/live cells/CD45 + /CD3 - /CD64 - F4/80 - /MHCII + CD11c + /CD11b + CD103 - . All CD103 + DC populations are singlets/live cells/CD45 + /CD3 - /CD64 - F4/80 - /MHCII + CD11c + /CD11b − CD103 + . All T cell populations are singlets/live cells/CD3 + /CD4 + . All correlations presented are Pearson’s correlations. Experimental schema was made with BioRender.com . Data in B-E are presented as mean ± SD. Data were analyzed in B–E using unpaired Student’s t-tests. Data are representative of 2 independent experiments. Statistical significance p value key is the following: ∗ = ≤ 0.05, ∗∗ = ≤ 0.01. See also Figure S11 .

Article Snippet: BUV737 Rat Anti-Mouse DLL4 (Clone: 9A1.5) , BD , Catalog #: 748394; RRID: AB_2872813.

Techniques: Expressing, Infection, Mutagenesis, Ex Vivo, Flow Cytometry

Journal: iScience

Article Title: Mycobacterium tuberculosis impedes CD40-dependent notch signaling to restrict Th 17 polarization during infection

doi: 10.1016/j.isci.2022.104305

Figure Lengend Snippet: Hip1 impedes DLL4 expression on lung DCs via a CD40-dependent mechanism C57BL/6 (CD40 +/+ ) or CD40 −/− mice were infected via the aerosol route with a low-dose of Mtb or hip1 mutant. Following 2 weeks post-infection, mice were euthanized and lung ex vivo responses were measured using flow cytometry. (A) Fold of infected mice over uninfected mice (from the same mouse strain) for DLL4 + and DLL4 + Jagged1 + in the CD103 + DC population. (B) Fold over uninfected for cytokine-positive CD4 T cells. (C) Fold over uninfected for NOTCH receptor-positive CD4 T cells. All CD11b + DC populations are singlets/live cells/CD45 + /CD3 - /CD64 - F4/80 - /MHCII + CD11c + /CD11b + CD103 - . All CD103 + DC populations are singlets/live cells/CD45 + /CD3 - /CD64 - F4/80 - /MHCII + CD11c + /CD11b − CD103 + . All T cell populations are singlets/live cells/CD3 + /CD4 + . Data were analyzed using a one-way ANOVA with a correction for multiple comparisons. Data are presented as fold over uninfected mean ± SD. Data are representative of 2 independent experiments. Statistical significance p value key is the following: ∗ = ≤ 0.05, ∗∗ = ≤ 0.01, ∗∗∗ = ≤ 0.001.

Article Snippet: BUV737 Rat Anti-Mouse DLL4 (Clone: 9A1.5) , BD , Catalog #: 748394; RRID: AB_2872813.

Techniques: Expressing, Infection, Mutagenesis, Ex Vivo, Flow Cytometry

Journal: iScience

Article Title: Mycobacterium tuberculosis impedes CD40-dependent notch signaling to restrict Th 17 polarization during infection

doi: 10.1016/j.isci.2022.104305

Figure Lengend Snippet:

Article Snippet: BUV737 Rat Anti-Mouse DLL4 (Clone: 9A1.5) , BD , Catalog #: 748394; RRID: AB_2872813.

Techniques: Blocking Assay, Purification, Mutagenesis, Recombinant, Lysis, Staining, Sequencing, Electron Microscopy, Negative Control, SYBR Green Assay, Cell Isolation, Enzyme-linked Immunosorbent Assay, Software

Journal: iScience

Article Title: Mycobacterium tuberculosis impedes CD40-dependent notch signaling to restrict Th 17 polarization during infection

doi: 10.1016/j.isci.2022.104305

Figure Lengend Snippet: Engaging CD40 on Mtb-infected DCs augments pro-inflammatory cytokines and enhances expression of Notch ligand Dll4 BMDCs were infected with either media (UI) or Mtb H37Rv strain at an MOI of 1 in the presence or absence of 1 μg/mL of multimeric CD40L reagent (CD40LT). At designated time points, cellfree supernatants and RNA were collected to assay for cytokine secretion and mRNA transcript. (A) ELISA measurements of IL-6 and IL-12p40 in supernatants. (B and C) qPCR analysis of genes was standardized to the housekeeping gene GAPDH, analyzed using the ΔΔCt method, and presented as 2 -ΔΔCt . Data are presented as mean ± SD (a) or mean ± SEM (B and C). Data are representative of 3 independent experiments. Data were analyzed in (A) using a one-way ANOVA with a correction for multiple comparisons and (B and C) using a two-way ANOVA with a correction for multiple comparisons. Statistical significance p value key is the following: ∗ = ≤ 0.05, ∗∗∗∗ = ≤ 0.0001. See also Figures S1–S4 .

Article Snippet: To stain lung innate cells, the following surface stain antibodies were used: BUV395 Rat Anti-Mouse CD84 (clone: 1D3/CD84, BD), BUV496 Rat Anti-Mouse I-A/I-E (clone: 2G9, BD), BUV563 Hamster Anti-Mouse CD80 (clone: 16-10A1, BD), BUV661 Rat Anti-Mouse CD115 (clone: T38-320, BD), BUV737 Rat Anti-Mouse DLL4 (clone: 9A1.5, BD), BUV805 Rat Anti-Mouse F4/80 (clone: T45-2342, BD), BV421 Rat Anti-Mouse CD172a (clone: P84, BD), BV421 anti-mouse CD169 (clone: 3D6.112, Biolegend), BV480 Hamster Anti-Mouse CD103 (clone: 2E7, BD), BV570 anti-mouse CD3 (clone: 17A2, Biolegend), BV570 anti-mouse CD19 (clone: 6D5, Biolegend), BV650 anti-mouse/rat XCR1 (clone: ZET, Biolegend), BV711 anti-mouse CD11c (clone: N418, Biolegend), BV750 anti-mouse CD45 (clone: 30-F11, Biolegend), BV786 Mouse Anti-Mouse CD64 a/b (clone: ×54-5/7.1, BD), FITC anti-mouse Ly-6G (clone: 1A8, Biolegend), BB700 Rat Anti-Mouse CD124 (clone: mIL4R-M1, BD), PE anti-mouse Jagged1 (clone: HMJ1-29, Biolegend), PE/Cy5 anti-mouse CD3ε (clone: 145-2C11, Biolegend), PE-Cy7 anti-mouse/human CD11b (clone: M1/70, Biolegend), PE/Dazzle 594 anti-mouse Ly-6C (clone: HK1.4, Biolegend), PE-Cy5 anti-mouse CD24 (clone: M1/69, Biolegend), PE-Cy7 anti-mouse JAML (clone: 4/E10, Novus Biologicals), Alexa 647 Rat Anti-Mouse S100A9 (clone: 2B10, BD), and Alexa 700 anti-mouse/human CD11b (clone: M1/70, Biolegend).

Techniques: Infection, Expressing, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: Mycobacterium tuberculosis impedes CD40-dependent notch signaling to restrict Th 17 polarization during infection

doi: 10.1016/j.isci.2022.104305

Figure Lengend Snippet: Engaging CD40 on Mtb-stimulated DCs enhances surface expression of DLL4 and Jagged1 BMDCs were stimulated with either media (UI) or HK Mtb (MOI 30) with or without the addition of 1 μg/mL of CD40LT. At designated time points, cells were collected and stained for surface markers. (A) Representative flow cytometry plot of DLL4 + and Jagged1 + frequencies. (B) Frequency of DLL4 + and Jagged1 + and double-positive populations. (C) MFI of DLL4 and Jagged1 expression and representative graphs. All populations are singlets/live cells/CD11c + MHCII hi . UI presented is 0H UI. Data are presented as mean ± SD. Data are representative of 3 independent experiments. Data were analyzed in (B) using a two-way ANOVA with a correction for multiple comparisons and (C) using an unpaired Student’s t test. Statistical significance p value key is the following: ∗ = ≤ 0.05, ∗∗ = ≤ 0.01, ∗∗∗ = ≤ 0.001, ∗∗∗∗ = ≤ 0.0001.

Article Snippet: To stain lung innate cells, the following surface stain antibodies were used: BUV395 Rat Anti-Mouse CD84 (clone: 1D3/CD84, BD), BUV496 Rat Anti-Mouse I-A/I-E (clone: 2G9, BD), BUV563 Hamster Anti-Mouse CD80 (clone: 16-10A1, BD), BUV661 Rat Anti-Mouse CD115 (clone: T38-320, BD), BUV737 Rat Anti-Mouse DLL4 (clone: 9A1.5, BD), BUV805 Rat Anti-Mouse F4/80 (clone: T45-2342, BD), BV421 Rat Anti-Mouse CD172a (clone: P84, BD), BV421 anti-mouse CD169 (clone: 3D6.112, Biolegend), BV480 Hamster Anti-Mouse CD103 (clone: 2E7, BD), BV570 anti-mouse CD3 (clone: 17A2, Biolegend), BV570 anti-mouse CD19 (clone: 6D5, Biolegend), BV650 anti-mouse/rat XCR1 (clone: ZET, Biolegend), BV711 anti-mouse CD11c (clone: N418, Biolegend), BV750 anti-mouse CD45 (clone: 30-F11, Biolegend), BV786 Mouse Anti-Mouse CD64 a/b (clone: ×54-5/7.1, BD), FITC anti-mouse Ly-6G (clone: 1A8, Biolegend), BB700 Rat Anti-Mouse CD124 (clone: mIL4R-M1, BD), PE anti-mouse Jagged1 (clone: HMJ1-29, Biolegend), PE/Cy5 anti-mouse CD3ε (clone: 145-2C11, Biolegend), PE-Cy7 anti-mouse/human CD11b (clone: M1/70, Biolegend), PE/Dazzle 594 anti-mouse Ly-6C (clone: HK1.4, Biolegend), PE-Cy5 anti-mouse CD24 (clone: M1/69, Biolegend), PE-Cy7 anti-mouse JAML (clone: 4/E10, Novus Biologicals), Alexa 647 Rat Anti-Mouse S100A9 (clone: 2B10, BD), and Alexa 700 anti-mouse/human CD11b (clone: M1/70, Biolegend).

Techniques: Expressing, Staining, Flow Cytometry

Journal: iScience

Article Title: Mycobacterium tuberculosis impedes CD40-dependent notch signaling to restrict Th 17 polarization during infection

doi: 10.1016/j.isci.2022.104305

Figure Lengend Snippet: DLL4 is required for Th 17 polarization but is dispensable for Th 1 polarization BMDCs were stimulated with either media alone (UI), or HK Mtb (MOI 30) with or without 1 μg/mL of CD40LT. Following 24H of stimulation, cells were pulsed with 10 μg/mL of cognate peptide (OVA 323-339 ) for one hour and then co-cultured with purified naïve CD4 OT-II Tg Thy1.1 T cells at a ratio of 4:1 T cells to DCs. For blockade conditions, blocking antibodies to either DLL4, Jagged1, or both DLL4 and Jagged1 combined, were added at the time of co-culture at the following concentrations: 15, 30, or 60 μg/mL. After 72H of co-culture, cell-free supernatants were harvested and assayed for cytokines by ELISA. (A) IFN-γ (Th 1 ). (B) IL-17 (Th 17 ). (C) IL-2. Data are presented as mean ± SD. Data are representative of 3 independent experiments. Data were analyzed using a one-way ANOVA with a correction for multiple comparisons (all data points were compared to Mtb + CD40LT). Statistical significance p value key is the following: ∗∗∗ = ≤ 0.001, ∗∗∗∗ = ≤ 0.0001. See also Figures S5–S7 .

Article Snippet: To stain lung innate cells, the following surface stain antibodies were used: BUV395 Rat Anti-Mouse CD84 (clone: 1D3/CD84, BD), BUV496 Rat Anti-Mouse I-A/I-E (clone: 2G9, BD), BUV563 Hamster Anti-Mouse CD80 (clone: 16-10A1, BD), BUV661 Rat Anti-Mouse CD115 (clone: T38-320, BD), BUV737 Rat Anti-Mouse DLL4 (clone: 9A1.5, BD), BUV805 Rat Anti-Mouse F4/80 (clone: T45-2342, BD), BV421 Rat Anti-Mouse CD172a (clone: P84, BD), BV421 anti-mouse CD169 (clone: 3D6.112, Biolegend), BV480 Hamster Anti-Mouse CD103 (clone: 2E7, BD), BV570 anti-mouse CD3 (clone: 17A2, Biolegend), BV570 anti-mouse CD19 (clone: 6D5, Biolegend), BV650 anti-mouse/rat XCR1 (clone: ZET, Biolegend), BV711 anti-mouse CD11c (clone: N418, Biolegend), BV750 anti-mouse CD45 (clone: 30-F11, Biolegend), BV786 Mouse Anti-Mouse CD64 a/b (clone: ×54-5/7.1, BD), FITC anti-mouse Ly-6G (clone: 1A8, Biolegend), BB700 Rat Anti-Mouse CD124 (clone: mIL4R-M1, BD), PE anti-mouse Jagged1 (clone: HMJ1-29, Biolegend), PE/Cy5 anti-mouse CD3ε (clone: 145-2C11, Biolegend), PE-Cy7 anti-mouse/human CD11b (clone: M1/70, Biolegend), PE/Dazzle 594 anti-mouse Ly-6C (clone: HK1.4, Biolegend), PE-Cy5 anti-mouse CD24 (clone: M1/69, Biolegend), PE-Cy7 anti-mouse JAML (clone: 4/E10, Novus Biologicals), Alexa 647 Rat Anti-Mouse S100A9 (clone: 2B10, BD), and Alexa 700 anti-mouse/human CD11b (clone: M1/70, Biolegend).

Techniques: Cell Culture, Purification, Blocking Assay, Co-Culture Assay, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: Mycobacterium tuberculosis impedes CD40-dependent notch signaling to restrict Th 17 polarization during infection

doi: 10.1016/j.isci.2022.104305

Figure Lengend Snippet: Blocking DLL4 singly or in combination with Jagged1 on DCs reduces antigen-specific IL-17 + CD4 T cell frequencies in vivo (A) Experimental schema. BMDCs we stimulated with either media alone (UI), or HK Mtb (MOI 30) with or without 1 μg/mL of CD40LT for 24H. At this time in the CD40LT condition, either DLL4 or DLL4 and Jagged1 blocking antibodies (30 μg/mL) were added. One day before transfer, 1E6 ESAT-6 transgenic T cells were transferred into mice via the intravenous (IV) route. On the day of intratracheal (IT) transfer, 1E6 DCs were transferred. At 6 days after transfer, mice were euthanized and lung single cell suspensions were stimulated with 10 μg/mL ESAT-6 1-20 peptide to assess antigen-specific responses. Cells were then stained for flow cytometry. (B) Representative flow cytometry plots of IFN-γ + and IL-17 + frequencies. (C) Cytokine-positive frequency of CD4 T cells. Populations shown are singlets/live cells/CD3 + /CD4 + . Experimental schema was made with BioRender.com . Data are presented as mean ± SD. Data are representative of 2 independent experiments. Data were analyzed using a one-way ANOVA with a correction for multiple comparisons. Statistical significance p value key is the following: ns = no significance, ∗ = ≤ 0.05, ∗∗ = ≤ 0.01, ∗∗∗ = ≤ 0.001, ∗∗∗∗ = ≤ 0.0001.

Article Snippet: To stain lung innate cells, the following surface stain antibodies were used: BUV395 Rat Anti-Mouse CD84 (clone: 1D3/CD84, BD), BUV496 Rat Anti-Mouse I-A/I-E (clone: 2G9, BD), BUV563 Hamster Anti-Mouse CD80 (clone: 16-10A1, BD), BUV661 Rat Anti-Mouse CD115 (clone: T38-320, BD), BUV737 Rat Anti-Mouse DLL4 (clone: 9A1.5, BD), BUV805 Rat Anti-Mouse F4/80 (clone: T45-2342, BD), BV421 Rat Anti-Mouse CD172a (clone: P84, BD), BV421 anti-mouse CD169 (clone: 3D6.112, Biolegend), BV480 Hamster Anti-Mouse CD103 (clone: 2E7, BD), BV570 anti-mouse CD3 (clone: 17A2, Biolegend), BV570 anti-mouse CD19 (clone: 6D5, Biolegend), BV650 anti-mouse/rat XCR1 (clone: ZET, Biolegend), BV711 anti-mouse CD11c (clone: N418, Biolegend), BV750 anti-mouse CD45 (clone: 30-F11, Biolegend), BV786 Mouse Anti-Mouse CD64 a/b (clone: ×54-5/7.1, BD), FITC anti-mouse Ly-6G (clone: 1A8, Biolegend), BB700 Rat Anti-Mouse CD124 (clone: mIL4R-M1, BD), PE anti-mouse Jagged1 (clone: HMJ1-29, Biolegend), PE/Cy5 anti-mouse CD3ε (clone: 145-2C11, Biolegend), PE-Cy7 anti-mouse/human CD11b (clone: M1/70, Biolegend), PE/Dazzle 594 anti-mouse Ly-6C (clone: HK1.4, Biolegend), PE-Cy5 anti-mouse CD24 (clone: M1/69, Biolegend), PE-Cy7 anti-mouse JAML (clone: 4/E10, Novus Biologicals), Alexa 647 Rat Anti-Mouse S100A9 (clone: 2B10, BD), and Alexa 700 anti-mouse/human CD11b (clone: M1/70, Biolegend).

Techniques: Blocking Assay, In Vivo, Transgenic Assay, Staining, Flow Cytometry

Journal: iScience

Article Title: Mycobacterium tuberculosis impedes CD40-dependent notch signaling to restrict Th 17 polarization during infection

doi: 10.1016/j.isci.2022.104305

Figure Lengend Snippet: Blocking DLL4 on Mtb-infected DCs reduces Th 17 and multifunctional CD4 T cell responses in vivo (A) Experimental schema. BMDCs were infected with Mtb at an MOI of 1, with or without CD40LT, for 48H. For antibody blockade studies, 60 μg/mL anti-DLL4 antibody was added during infection. DCs were then harvested and 1E6 were intratracheally (IT) transferred into the lungs of mice along with additional blocking antibody. At 4 weeks post transfer, mice were euthanized and lung suspensions were unstimulated to asses ex vivo responses using flow cytometry. (B) Frequency of cytokine-positive CD4 T cells. (C) Boolean analysis of frequency of multiple cytokine-positive CD4 T cells. (D) Frequency of CXCR3 + CCR6 + CD4 T cells and Boolean analysis of CXCR3 + CCR6 + and cytokine-positive CD4 T cells. All populations are singlets/live cells/CD3 + /CD4 + . Experimental schema was made with BioRender.com . Data are presented as mean ± SD. Data are representative of 2 independent experiments. Data were analyzed using a one-way ANOVA with a correction for multiple comparisons. Statistical significance p value key is the following: ∗ = ≤ 0.05, ∗∗ = ≤ 0.01, ∗∗∗ = ≤ 0.001. SP = single-positive, DP = double-positive, TP = triple-positive. See also Figures S8 and .

Article Snippet: To stain lung innate cells, the following surface stain antibodies were used: BUV395 Rat Anti-Mouse CD84 (clone: 1D3/CD84, BD), BUV496 Rat Anti-Mouse I-A/I-E (clone: 2G9, BD), BUV563 Hamster Anti-Mouse CD80 (clone: 16-10A1, BD), BUV661 Rat Anti-Mouse CD115 (clone: T38-320, BD), BUV737 Rat Anti-Mouse DLL4 (clone: 9A1.5, BD), BUV805 Rat Anti-Mouse F4/80 (clone: T45-2342, BD), BV421 Rat Anti-Mouse CD172a (clone: P84, BD), BV421 anti-mouse CD169 (clone: 3D6.112, Biolegend), BV480 Hamster Anti-Mouse CD103 (clone: 2E7, BD), BV570 anti-mouse CD3 (clone: 17A2, Biolegend), BV570 anti-mouse CD19 (clone: 6D5, Biolegend), BV650 anti-mouse/rat XCR1 (clone: ZET, Biolegend), BV711 anti-mouse CD11c (clone: N418, Biolegend), BV750 anti-mouse CD45 (clone: 30-F11, Biolegend), BV786 Mouse Anti-Mouse CD64 a/b (clone: ×54-5/7.1, BD), FITC anti-mouse Ly-6G (clone: 1A8, Biolegend), BB700 Rat Anti-Mouse CD124 (clone: mIL4R-M1, BD), PE anti-mouse Jagged1 (clone: HMJ1-29, Biolegend), PE/Cy5 anti-mouse CD3ε (clone: 145-2C11, Biolegend), PE-Cy7 anti-mouse/human CD11b (clone: M1/70, Biolegend), PE/Dazzle 594 anti-mouse Ly-6C (clone: HK1.4, Biolegend), PE-Cy5 anti-mouse CD24 (clone: M1/69, Biolegend), PE-Cy7 anti-mouse JAML (clone: 4/E10, Novus Biologicals), Alexa 647 Rat Anti-Mouse S100A9 (clone: 2B10, BD), and Alexa 700 anti-mouse/human CD11b (clone: M1/70, Biolegend).

Techniques: Blocking Assay, Infection, In Vivo, Ex Vivo, Flow Cytometry

Journal: iScience

Article Title: Mycobacterium tuberculosis impedes CD40-dependent notch signaling to restrict Th 17 polarization during infection

doi: 10.1016/j.isci.2022.104305

Figure Lengend Snippet: Engaging CD40 on Mtb-infected DCs increases NOTCH2 expression on CD4 T cells in the lung BMDCs were infected with Mtb at an MOI of 1, with or without CD40LT, for 48H. For antibody blockade studies, 60 μg/mL anti-DLL4 antibody was added during infection. DCs were then harvested and 1E6 were intratracheally (IT) transferred into the lungs of mice along with additional blocking antibody. At 4 weeks post transfer, mice were euthanized and lung suspensions were unstimulated to asses ex vivo responses using flow cytometry. (A) Frequency of NOTCH receptor-positive CD4 T cells. (B) Boolean analysis of NOTCH receptor-expressing and cytokine-positive CD4 T cells. All populations are singlets/live cells/CD3 + /CD4 + . Data are presented as mean ± SD. Data are representative of 2 independent experiments. Data were analyzed using a one-way ANOVA with a correction for multiple comparisons. Statistical significance p value key is the following: ∗ = ≤ 0.05, ∗∗ = ≤ 0.01, ∗∗∗ = ≤ 0.001, ∗∗∗∗ = ≤ 0.0001.

Article Snippet: To stain lung innate cells, the following surface stain antibodies were used: BUV395 Rat Anti-Mouse CD84 (clone: 1D3/CD84, BD), BUV496 Rat Anti-Mouse I-A/I-E (clone: 2G9, BD), BUV563 Hamster Anti-Mouse CD80 (clone: 16-10A1, BD), BUV661 Rat Anti-Mouse CD115 (clone: T38-320, BD), BUV737 Rat Anti-Mouse DLL4 (clone: 9A1.5, BD), BUV805 Rat Anti-Mouse F4/80 (clone: T45-2342, BD), BV421 Rat Anti-Mouse CD172a (clone: P84, BD), BV421 anti-mouse CD169 (clone: 3D6.112, Biolegend), BV480 Hamster Anti-Mouse CD103 (clone: 2E7, BD), BV570 anti-mouse CD3 (clone: 17A2, Biolegend), BV570 anti-mouse CD19 (clone: 6D5, Biolegend), BV650 anti-mouse/rat XCR1 (clone: ZET, Biolegend), BV711 anti-mouse CD11c (clone: N418, Biolegend), BV750 anti-mouse CD45 (clone: 30-F11, Biolegend), BV786 Mouse Anti-Mouse CD64 a/b (clone: ×54-5/7.1, BD), FITC anti-mouse Ly-6G (clone: 1A8, Biolegend), BB700 Rat Anti-Mouse CD124 (clone: mIL4R-M1, BD), PE anti-mouse Jagged1 (clone: HMJ1-29, Biolegend), PE/Cy5 anti-mouse CD3ε (clone: 145-2C11, Biolegend), PE-Cy7 anti-mouse/human CD11b (clone: M1/70, Biolegend), PE/Dazzle 594 anti-mouse Ly-6C (clone: HK1.4, Biolegend), PE-Cy5 anti-mouse CD24 (clone: M1/69, Biolegend), PE-Cy7 anti-mouse JAML (clone: 4/E10, Novus Biologicals), Alexa 647 Rat Anti-Mouse S100A9 (clone: 2B10, BD), and Alexa 700 anti-mouse/human CD11b (clone: M1/70, Biolegend).

Techniques: Infection, Expressing, Blocking Assay, Ex Vivo, Flow Cytometry

Journal: iScience

Article Title: Mycobacterium tuberculosis impedes CD40-dependent notch signaling to restrict Th 17 polarization during infection

doi: 10.1016/j.isci.2022.104305

Figure Lengend Snippet: Th 17 responses correlate with NOTCH2 expression and lower lung CFU BMDCs were infected with Mtb at an MOI of 1, with or without CD40LT, for 48H. For antibody blockade studies, 60 μg/mL anti-DLL4 antibody was added during infection. DCs were then harvested and 1E6 were intratracheally (IT) transferred into the lungs of mice along with additional blocking antibody. At 4 weeks post transfer, mice were euthanized and lung suspensions were unstimulated to asses ex vivo responses using flow cytometry and lung homogenates were plated to enumerate Mtb burdens. (A) Correlogram using R package “corrplot” showing correlation between the frequency of marker-positive CD4 T cells and frequency of cytokine-positive CD4 T cells. (B) Correlations in R using “ggscatter” between the frequency of marker-positive CD4 T cells and frequency of cytokine-positive CD4 T cells. (C) Mtb lung colony-forming unit (CFU) and correlations between CFU and frequencies of marker-positive or cytokine-positive CD4 T cells using “ggscatter” in R. All correlations presented are Pearson’s correlations. Data in C) are presented as mean ± SD and were analyzed using an unpaired Student’s t test. Data are representative of 2 independent experiments. Statistical significance p value key is the following: ∗ = ≤ 0.05, ∗∗ = ≤ 0.01, ∗∗∗ = ≤ 0.001, ∗∗∗∗ = ≤ 0.0001. See also Figure S10 .

Article Snippet: To stain lung innate cells, the following surface stain antibodies were used: BUV395 Rat Anti-Mouse CD84 (clone: 1D3/CD84, BD), BUV496 Rat Anti-Mouse I-A/I-E (clone: 2G9, BD), BUV563 Hamster Anti-Mouse CD80 (clone: 16-10A1, BD), BUV661 Rat Anti-Mouse CD115 (clone: T38-320, BD), BUV737 Rat Anti-Mouse DLL4 (clone: 9A1.5, BD), BUV805 Rat Anti-Mouse F4/80 (clone: T45-2342, BD), BV421 Rat Anti-Mouse CD172a (clone: P84, BD), BV421 anti-mouse CD169 (clone: 3D6.112, Biolegend), BV480 Hamster Anti-Mouse CD103 (clone: 2E7, BD), BV570 anti-mouse CD3 (clone: 17A2, Biolegend), BV570 anti-mouse CD19 (clone: 6D5, Biolegend), BV650 anti-mouse/rat XCR1 (clone: ZET, Biolegend), BV711 anti-mouse CD11c (clone: N418, Biolegend), BV750 anti-mouse CD45 (clone: 30-F11, Biolegend), BV786 Mouse Anti-Mouse CD64 a/b (clone: ×54-5/7.1, BD), FITC anti-mouse Ly-6G (clone: 1A8, Biolegend), BB700 Rat Anti-Mouse CD124 (clone: mIL4R-M1, BD), PE anti-mouse Jagged1 (clone: HMJ1-29, Biolegend), PE/Cy5 anti-mouse CD3ε (clone: 145-2C11, Biolegend), PE-Cy7 anti-mouse/human CD11b (clone: M1/70, Biolegend), PE/Dazzle 594 anti-mouse Ly-6C (clone: HK1.4, Biolegend), PE-Cy5 anti-mouse CD24 (clone: M1/69, Biolegend), PE-Cy7 anti-mouse JAML (clone: 4/E10, Novus Biologicals), Alexa 647 Rat Anti-Mouse S100A9 (clone: 2B10, BD), and Alexa 700 anti-mouse/human CD11b (clone: M1/70, Biolegend).

Techniques: Expressing, Infection, Blocking Assay, Ex Vivo, Flow Cytometry, Marker

Journal: iScience

Article Title: Mycobacterium tuberculosis impedes CD40-dependent notch signaling to restrict Th 17 polarization during infection

doi: 10.1016/j.isci.2022.104305

Figure Lengend Snippet: Mtb restricts DLL4 expression and early CD4 T cells responses in the lung through the Hip1 serine protease (A) Experimental schema. C57BL/6 mice were infected via the aerosol route with a low-dose of Mtb or hip1 mutant. Following 2 weeks post-infection, mice were euthanized and lung ex vivo responses were measured using flow cytometry. (B–D) (B) Frequency of CD40-expressing CD11b + DCs and CD40-expressing CD103 + DCs. Representative flow plots and frequency of DLL4 + , Jagged1 + , and DLL4 + Jagged1 + for (C) CD11b + DCs and (D) CD103 + DCs. (E) Frequency of cytokine-positive and NOTCH receptor-positive CD4 T cells. (F) Correlations between different CD4 T cell and innate immune population markers using “ggscatter” in R. All CD11b + DC populations are singlets/live cells/CD45 + /CD3 - /CD64 - F4/80 - /MHCII + CD11c + /CD11b + CD103 - . All CD103 + DC populations are singlets/live cells/CD45 + /CD3 - /CD64 - F4/80 - /MHCII + CD11c + /CD11b − CD103 + . All T cell populations are singlets/live cells/CD3 + /CD4 + . All correlations presented are Pearson’s correlations. Experimental schema was made with BioRender.com . Data in B-E are presented as mean ± SD. Data were analyzed in B–E using unpaired Student’s t-tests. Data are representative of 2 independent experiments. Statistical significance p value key is the following: ∗ = ≤ 0.05, ∗∗ = ≤ 0.01. See also Figure S11 .

Article Snippet: To stain lung innate cells, the following surface stain antibodies were used: BUV395 Rat Anti-Mouse CD84 (clone: 1D3/CD84, BD), BUV496 Rat Anti-Mouse I-A/I-E (clone: 2G9, BD), BUV563 Hamster Anti-Mouse CD80 (clone: 16-10A1, BD), BUV661 Rat Anti-Mouse CD115 (clone: T38-320, BD), BUV737 Rat Anti-Mouse DLL4 (clone: 9A1.5, BD), BUV805 Rat Anti-Mouse F4/80 (clone: T45-2342, BD), BV421 Rat Anti-Mouse CD172a (clone: P84, BD), BV421 anti-mouse CD169 (clone: 3D6.112, Biolegend), BV480 Hamster Anti-Mouse CD103 (clone: 2E7, BD), BV570 anti-mouse CD3 (clone: 17A2, Biolegend), BV570 anti-mouse CD19 (clone: 6D5, Biolegend), BV650 anti-mouse/rat XCR1 (clone: ZET, Biolegend), BV711 anti-mouse CD11c (clone: N418, Biolegend), BV750 anti-mouse CD45 (clone: 30-F11, Biolegend), BV786 Mouse Anti-Mouse CD64 a/b (clone: ×54-5/7.1, BD), FITC anti-mouse Ly-6G (clone: 1A8, Biolegend), BB700 Rat Anti-Mouse CD124 (clone: mIL4R-M1, BD), PE anti-mouse Jagged1 (clone: HMJ1-29, Biolegend), PE/Cy5 anti-mouse CD3ε (clone: 145-2C11, Biolegend), PE-Cy7 anti-mouse/human CD11b (clone: M1/70, Biolegend), PE/Dazzle 594 anti-mouse Ly-6C (clone: HK1.4, Biolegend), PE-Cy5 anti-mouse CD24 (clone: M1/69, Biolegend), PE-Cy7 anti-mouse JAML (clone: 4/E10, Novus Biologicals), Alexa 647 Rat Anti-Mouse S100A9 (clone: 2B10, BD), and Alexa 700 anti-mouse/human CD11b (clone: M1/70, Biolegend).

Techniques: Expressing, Infection, Mutagenesis, Ex Vivo, Flow Cytometry

Journal: iScience

Article Title: Mycobacterium tuberculosis impedes CD40-dependent notch signaling to restrict Th 17 polarization during infection

doi: 10.1016/j.isci.2022.104305

Figure Lengend Snippet: Hip1 impedes DLL4 expression on lung DCs via a CD40-dependent mechanism C57BL/6 (CD40 +/+ ) or CD40 −/− mice were infected via the aerosol route with a low-dose of Mtb or hip1 mutant. Following 2 weeks post-infection, mice were euthanized and lung ex vivo responses were measured using flow cytometry. (A) Fold of infected mice over uninfected mice (from the same mouse strain) for DLL4 + and DLL4 + Jagged1 + in the CD103 + DC population. (B) Fold over uninfected for cytokine-positive CD4 T cells. (C) Fold over uninfected for NOTCH receptor-positive CD4 T cells. All CD11b + DC populations are singlets/live cells/CD45 + /CD3 - /CD64 - F4/80 - /MHCII + CD11c + /CD11b + CD103 - . All CD103 + DC populations are singlets/live cells/CD45 + /CD3 - /CD64 - F4/80 - /MHCII + CD11c + /CD11b − CD103 + . All T cell populations are singlets/live cells/CD3 + /CD4 + . Data were analyzed using a one-way ANOVA with a correction for multiple comparisons. Data are presented as fold over uninfected mean ± SD. Data are representative of 2 independent experiments. Statistical significance p value key is the following: ∗ = ≤ 0.05, ∗∗ = ≤ 0.01, ∗∗∗ = ≤ 0.001.

Article Snippet: To stain lung innate cells, the following surface stain antibodies were used: BUV395 Rat Anti-Mouse CD84 (clone: 1D3/CD84, BD), BUV496 Rat Anti-Mouse I-A/I-E (clone: 2G9, BD), BUV563 Hamster Anti-Mouse CD80 (clone: 16-10A1, BD), BUV661 Rat Anti-Mouse CD115 (clone: T38-320, BD), BUV737 Rat Anti-Mouse DLL4 (clone: 9A1.5, BD), BUV805 Rat Anti-Mouse F4/80 (clone: T45-2342, BD), BV421 Rat Anti-Mouse CD172a (clone: P84, BD), BV421 anti-mouse CD169 (clone: 3D6.112, Biolegend), BV480 Hamster Anti-Mouse CD103 (clone: 2E7, BD), BV570 anti-mouse CD3 (clone: 17A2, Biolegend), BV570 anti-mouse CD19 (clone: 6D5, Biolegend), BV650 anti-mouse/rat XCR1 (clone: ZET, Biolegend), BV711 anti-mouse CD11c (clone: N418, Biolegend), BV750 anti-mouse CD45 (clone: 30-F11, Biolegend), BV786 Mouse Anti-Mouse CD64 a/b (clone: ×54-5/7.1, BD), FITC anti-mouse Ly-6G (clone: 1A8, Biolegend), BB700 Rat Anti-Mouse CD124 (clone: mIL4R-M1, BD), PE anti-mouse Jagged1 (clone: HMJ1-29, Biolegend), PE/Cy5 anti-mouse CD3ε (clone: 145-2C11, Biolegend), PE-Cy7 anti-mouse/human CD11b (clone: M1/70, Biolegend), PE/Dazzle 594 anti-mouse Ly-6C (clone: HK1.4, Biolegend), PE-Cy5 anti-mouse CD24 (clone: M1/69, Biolegend), PE-Cy7 anti-mouse JAML (clone: 4/E10, Novus Biologicals), Alexa 647 Rat Anti-Mouse S100A9 (clone: 2B10, BD), and Alexa 700 anti-mouse/human CD11b (clone: M1/70, Biolegend).

Techniques: Expressing, Infection, Mutagenesis, Ex Vivo, Flow Cytometry

Journal: iScience

Article Title: Mycobacterium tuberculosis impedes CD40-dependent notch signaling to restrict Th 17 polarization during infection

doi: 10.1016/j.isci.2022.104305

Figure Lengend Snippet:

Article Snippet: To stain lung innate cells, the following surface stain antibodies were used: BUV395 Rat Anti-Mouse CD84 (clone: 1D3/CD84, BD), BUV496 Rat Anti-Mouse I-A/I-E (clone: 2G9, BD), BUV563 Hamster Anti-Mouse CD80 (clone: 16-10A1, BD), BUV661 Rat Anti-Mouse CD115 (clone: T38-320, BD), BUV737 Rat Anti-Mouse DLL4 (clone: 9A1.5, BD), BUV805 Rat Anti-Mouse F4/80 (clone: T45-2342, BD), BV421 Rat Anti-Mouse CD172a (clone: P84, BD), BV421 anti-mouse CD169 (clone: 3D6.112, Biolegend), BV480 Hamster Anti-Mouse CD103 (clone: 2E7, BD), BV570 anti-mouse CD3 (clone: 17A2, Biolegend), BV570 anti-mouse CD19 (clone: 6D5, Biolegend), BV650 anti-mouse/rat XCR1 (clone: ZET, Biolegend), BV711 anti-mouse CD11c (clone: N418, Biolegend), BV750 anti-mouse CD45 (clone: 30-F11, Biolegend), BV786 Mouse Anti-Mouse CD64 a/b (clone: ×54-5/7.1, BD), FITC anti-mouse Ly-6G (clone: 1A8, Biolegend), BB700 Rat Anti-Mouse CD124 (clone: mIL4R-M1, BD), PE anti-mouse Jagged1 (clone: HMJ1-29, Biolegend), PE/Cy5 anti-mouse CD3ε (clone: 145-2C11, Biolegend), PE-Cy7 anti-mouse/human CD11b (clone: M1/70, Biolegend), PE/Dazzle 594 anti-mouse Ly-6C (clone: HK1.4, Biolegend), PE-Cy5 anti-mouse CD24 (clone: M1/69, Biolegend), PE-Cy7 anti-mouse JAML (clone: 4/E10, Novus Biologicals), Alexa 647 Rat Anti-Mouse S100A9 (clone: 2B10, BD), and Alexa 700 anti-mouse/human CD11b (clone: M1/70, Biolegend).

Techniques: Blocking Assay, Purification, Mutagenesis, Recombinant, Lysis, Staining, Sequencing, Electron Microscopy, Negative Control, SYBR Green Assay, Cell Isolation, Enzyme-linked Immunosorbent Assay, Software