Journal: STAR Protocols

Article Title: Protocol to colonize gnotobiotic mice in early life and assess the impact on early life immune programming

doi: 10.1016/j.xpro.2022.101914

Figure Lengend Snippet:

Article Snippet: TNFα (Clone MP6-XT22), 40× , Miltenyi Biotec , Cat#130-102-386; RRID: AB_2661141.

Techniques: Recombinant, Extraction, Diagnostic Assay, Staining, cDNA Synthesis, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Software

Journal: Nature Immunology

Article Title: A spatially coordinated keratinocyte–fibroblast circuit recruits MMP9 + myeloid cells to drive type I interferon-driven inflammation in photosensitive autoimmunity

doi: 10.1038/s41590-026-02502-w

Figure Lengend Snippet: a , Keratinocyte viability, measured by propidium iodide (PI) uptake ( n = 6) and ATP depletion ( n = 8). Data are presented as means and were analyzed using one-way ANOVA followed by Bonferroni-corrected pairwise comparisons; error bars, s.e.m. A P value < 0.05 was considered statistically significant. b , Dot plots of moDC activation marker expression (CD83, CD86) following exposure to keratinocyte-conditioned media or direct IFNβ stimulation; lipopolysaccharide (LPS) was used as a positive control. Data are presented as means; error bars, s.e.m. Statistical analysis was performed using a one-way ANOVA followed by Bonferroni-corrected multiple comparisons. Each dot represents one biological replicate. Data were derived from six independent donors. c , Heatmap of differentially expressed genes ( P < 0.05, |log 2 (fold change)| > 1) in bulk RNA-seq of moDCs incubated with keratinocyte supernatants treated with UVB and IFNβ; data are counts per million (CPM) scaled by gene (row) ( n = 5 donors per group). d , Heatmap of differentially abundant protein expression (NPQ) ( P < 0.05) in moDCs after incubation with keratinocyte supernatants from the indicated conditions; colors scaled by protein (row) ( n = 4 donors per group). e , Heatmap of MMP9 + CD14 + signature genes found induced in moDCs after exposure to keratinocyte-conditioned media or direct IFNβ. Signature genes are M1 markers and M2 markers in Fig. that are high on the MMP9 end; data are counts per million scaled by gene (row). f , g , Percentage of moDCs expressing CD83 following stimulation with keratinocyte supernatants treated with proteinase K (PK), DNase or passed through a 0.22 μm filter. Data are presented as means; error bars, s.e.m. Statistical analysis was performed using a one-way ANOVA followed by Bonferroni-corrected multiple comparisons. Each dot represents one biological replicate. Data in f were derived from eight independent donors, and data in g were derived from six independent donors. h , Cytokine concentration in keratinocyte supernatants, measured using OLINK proteomics. Data are presented as means and were analyzed using one-way ANOVA followed by Tukey’s multiple comparison test; error bars, s.e.m. Each dot represents one independent donor. Data were derived from four independent donors. i , Percentage of moDCs expressing CD83 following incubation with keratinocyte supernatants pre-treated with TNF neutralizing antibody (adalimumab) or IFNAR blocker (anifrolumab) under UVB and IFNβ conditions. Data are presented as means; error bars, s.e.m. Statistical analysis was performed using a one-way ANOVA followed by Bonferroni-corrected multiple comparisons. Each dot represents one biological replicate. Data were derived from six independent donors. j , Heatmap of differentially expressed genes ( P < 0.05, |log 2 (fold change)| > 1) in fibroblasts treated with conditioned keratinocyte supernatants, and direct IFNβ, IFNγ, IL1A or TNF; data are counts per million scaled by gene (row). Data were derived from four independent donors. k , Number of CD4 + (left) and CD8 + (right) T cells by flow cytometry that migrated in Boyden chambers, recruited by supernatants of moDCs stimulated with keratinocyte supernatants (in color) or supernatants of moDCs stimulated with PBS and LPS as controls. Data are presented as means and were analyzed using one-way ANOVA followed by Tukey’s multiple comparison test; error bars, s.e.m. Each symbol represents one biological replicate. Experiments were performed using cells derived from four independent donors.

Article Snippet: For blocking assays, moDCs were pre-incubated with adalimumab (20 μg ml −1 , 1 h; HY-P9908, MedChemExpress) or anifrolumab (50 μg ml −1 , 24 h; HY- P99168 , MedChemExpress) before stimulation.

Techniques: Activation Assay, Marker, Expressing, Positive Control, Derivative Assay, RNA Sequencing, Incubation, Concentration Assay, Comparison, Flow Cytometry

Journal:

Article Title:

doi:

Figure Lengend Snippet: Melittin attenuates the expression of pro-inflammatory cytokine in obstructed kidneys. ( A ) Immunohistochemical staining shows that melittin inhibits the expressions of tumor necrosis factor (TNF)-α and interleukin (IL)-1β in kidneys at 7 days after unilateral ureteral obstruction (UUO) surgery. Immunohistochemical staining was used to evaluate the extent of pro-inflammatory cytokines, which was subsequently quantified. ( B , C ) Western blot analysis and Reverse transcription-polymerase chain reaction (RT-PCR) results show that melittin suppresses the protein and mRNA expressions of TNF-α and IL-1β in UUO kidneys. Glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) levels were analyzed as an internal control. −: not treated, +: treated. These are representative images from each study group. Magnification: 400×. The results are expressed as means ± SE of three independent determinations. * p < 0.05 vs. Sham group. † p < 0.05 vs. Vehicle group.

Article Snippet: The primary antibodies were follows: anti-TNF-α and anti-fibronectin (Abcam, Cambridge, MA, USA), anti-IL-1β (Santa Cruz Biotechnology, Dallas, TX, USA), and anti-TGF-β1 (R&D Systems, Minneapolis, MN, USA).

Techniques: Expressing, Immunohistochemical staining, Staining, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Frontiers in Veterinary Science

Article Title: Immunization of Cattle With Recombinant Structural Ectodomains I and II of Babesia bovis Apical Membrane Antigen 1 [BbAMA-1(I/II)] Induces Strong Th1 Immune Response

doi: 10.3389/fvets.2022.917389

Figure Lengend Snippet: Monoclonal and polyclonal antibodies (mAbs) used in this study for flow cytometry analysis and immunological assay.

Article Snippet: Anti-TNF alpha , Alexa Fluor® 488 , CC327 , Mouse , 1:200 , MCA2334A488 (AbD Serotec, United Kingdom).

Techniques: Flow Cytometry

Journal: Pediatric Research

Article Title: Lipopolysaccharide Preconditioning Reduces Neuroinflammation Against Hypoxic Ischemia and Provides Long-Term Outcome of Neuroprotection in Neonatal Rat

doi: 10.1203/pdr.0b013e3181b0d336

Figure Lengend Snippet: Figure 1. Immunohistochemistry for CD-11b and TNF-alpha per- formed 12 h after LPS injections. LPS activated few microglias in the cerebral cortex (A) 12-h postinjec- tion; however, there were signifi- cantly more CD-11b () cells (ar- rows) in the white matter (B) of rats treated with LPS0.3 than those treated with LPS0.05 or NS. TNF- alpha levels were significantly higher in the cerebral cortex (C) and white matter (D) in the LPS0.3 group than in the other two groups (IOD, inte- grated OD). Differences in TNF- alpha levels between the LPS0.05 and NS groups were not significant. Scale bar 50 m in (A, B, C) and 100 m in (D). Data are from three rats in each group. Values are means SEM. *p 0.05, and **p 0.01.

Article Snippet: The specific primary antibodies included anti-rat TNF-alpha antibody (1:100) (Cat. # BMS175; Bender MedSystems, Vienna, Austria) and anti-rat microglia and macrophage antibody (CD-11b; 1:100) (Cat. # MCA275R; AbD Serotec, Oxford, UK).

Techniques: Immunohistochemistry

Journal: Pediatric Research

Article Title: Lipopolysaccharide Preconditioning Reduces Neuroinflammation Against Hypoxic Ischemia and Provides Long-Term Outcome of Neuroprotection in Neonatal Rat

doi: 10.1203/pdr.0b013e3181b0d336

Figure Lengend Snippet: Figure 3. Immunohistochemistry for CD-11b and TNF-alpha per- formed 24 h after HI. The LPS-HI group had significantly reduced CD- 11b-() cells in the ipsilateral cortex (A) and white matter (B) than the NS-HI group. TNF-alpha level was also significantly decreased in the ce- rebral cortex (C) and white matter (D) in the LPS-HI group than in the NS-HI group. Data are from three rats in each group. Values are means SEM. Scale bar 50 m in (A-D). *p 0.05, **p 0.01, and §p 0.001.

Article Snippet: The specific primary antibodies included anti-rat TNF-alpha antibody (1:100) (Cat. # BMS175; Bender MedSystems, Vienna, Austria) and anti-rat microglia and macrophage antibody (CD-11b; 1:100) (Cat. # MCA275R; AbD Serotec, Oxford, UK).

Techniques: Immunohistochemistry