Journal: Bio-protocol

Article Title: Nuclei Isolation Methods on Frozen Clotted Blood Samples

doi: 10.21769/BioProtoc.5573

Figure Lengend Snippet: (A) Gating strategy for sorting singlets of B and T cells. (B) Sorted counts. Leukocytes were isolated from previously frozen blood clots and dissociated by following section A of this protocol. Cells were stained with an antibody panel targeting CD3, CD19, CD11b, and CD11c. Viability marker was not used due to interest in nuclei from subpopulations of non-viable leukocytes. After doublet exclusion and gating on height and area of cells, T-cell subsets were identified as CD3+ populations. B-cell subsets were identified as CD19+ populations. CD11b and CD11c were used to exclude monocytes. Single-stain controls were used for compensation. During this sort, we successfully sorted 57,894 T cells and 24,648 B cells. The full report is available in Supplementary material (Report S1).

Article Snippet: Flow cytometry antibodies (only applicable if flow sorting) a. FITC CD3 antibody (Miltenyi Biotec, catalog number: 130-113-700) b. PE CD19 antibody (Miltenyi Biotec, catalog number: 130-114-172) c. VioBlue CD11B (Miltenyi Biotec, catalog number: 130-110-616) d. APC CD11C (Miltenyi Biotec, catalog number: 130-114-110) e. Other antibodies (optional): Add other suitable antibodies to tailor the cell population of interest 3.

Techniques: Isolation, Staining, Marker

Journal: Frontiers in Immunology

Article Title: Sustained STING-IRF7 signaling aggravates LPS-induced endometrial inflammation via excessive neutrophil extracellular traps generation

doi: 10.3389/fimmu.2025.1671848

Figure Lengend Snippet: CD11b was regulated by STING-IRF7 pathway in endometrium. (A) Representative immunohistochemical staining of CD11b in endometrial tissues of WT or STING-deficient mice ( Tmem173 gt ) infected by LPS for 24 h at 400 × magnification (scale bar = 25 μm). (B) The area of CD11b were quantified (n=6). One-way ANOVA test was applied with ** P <0.01 (WT vs. Tmem173 gt in LPS group). (C) STING inhibitor H-151was used to pretreat Ishikawa cell with or without LPS infected for 24 h, then the protein expression of CD11b was detected by Western blotting. (D) Quantification of the amount of CD11b in the four groups (n=5). One-way ANOVA test was applied with * P <0.05 (LPS vs. H-151+LPS group). (E) Quantitative mRNA expression of irf7 in endometrium of mice (n=6). One-way ANOVA test was applied with * P <0.05 (WT vs. Tmem173 gt in LPS group) (F) Representative immunoblots of IRF7 in LPS-stimulated STING-deficient mice ( Tmem173 gt ) endometrium tissues, compared with WT-LPS stimulated mice. (G) Quantification of the amount of IRF7 in the four groups (n=4). One-way ANOVA test was applied with **** P <0.0001(WT vs. Tmem173 gt in LPS group). (H) Representative immunoblots of CEBPB in LPS-stimulated STING-deficient mice ( Tmem173 gt ) endometrium tissues, compared with WT-LPS stimulated mice. (I) Representative immunoblots of IRF7 in STING inhibitor H-151 pretreat Ishikawa cell. (J) Quantification of the amount of IRF7 in the two groups, and t test was applied with * P <0.05 (LPS vs. H-151+LPS group). (K) Quantitative mRNA expression of itgam with IRF7 overexpression in Ishikawa cells, and t test was applied with *** P <0.001 (LPS vs. H-151+LPS group). (L) Representative immunoblots of CD11b with the transfection of IRF7 in Ishikawa cells. (M) Quantification of the amount of CD11b in the two groups, and t test was applied with * P <0.05 (LPS vs. IRF7+LPS group). (N) Detection of CD11b expression by flow cytometry in mice bone marrow neutrophils stimulated with STING inhibitor C-176. (O) Quantification of CD11b expression on Ly6G + CD45 + cells in mice bone marrow neutrophils stimulated with STING inhibitor C-176., and t test was applied with **** P <0.0001. Independent experiments are repeated at least three times.

Article Snippet: After blocking with 5% non-fat milk for 1 h, cells were incubated with the following antibodies: IL-1β (1:1000, 26048-1-AP, Proteintech, China), citH3 (1:1000, AB281584, Abcam, USA), ELA2 (1:1000, 27642-1-AP, Proteintech, China) and MPO (1:1000, 22225-1-AP, Proteintech, China), CD11b (MAC-1) (1:1000, AB133357, Abcam, MA, USA), IRF7(1:1000, Cat. no. 22392-1-AP, Proteintech, Wuhan, China), CEBPB (1:1000, D155298; BBI, China), and VDR (1:1000, D151709, BBI, China), and GAPDH (1:1000, 60004-1-AP; Proteintech, China) overnight at 4°C and washed in Tris-buffered saline (TBST) in triplicate for 5 min.

Techniques: Immunohistochemical staining, Staining, Infection, Expressing, Western Blot, Over Expression, Transfection, Flow Cytometry