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: PloS one

Article Title: Phagocytosis is the main CR3-mediated function affected by the lupus-associated variant of CD11b in human myeloid cells.

doi: 10.1371/journal.pone.0057082

Figure Lengend Snippet: Figure 1. Cell surface expression of CD11b on different cell populations. The expression was quantified by flow cytometry using ICRF44 (A) and CBRM1/5 (B) antibodies. The latter only recognises the headpiece of CD11b in its active state. Data are presented in mean fluorescence intensity (MFI), closed symbols 77R/R donors, open symbols 77R/H-77H/H donors. The two groups were not statistically different. Bars indicate means. doi:10.1371/journal.pone.0057082.g001

Article Snippet: The cDNA of the 77R CD11b allele was obtained from OriGene Technologies (Rockville, MD, USA, SC315229, NM000632.2), whilst the lupus susceptible allele (77H) was obtained by site-directed mutagenesis by changing the G at position 230 to an A (R77H).

Techniques: Expressing, Flow Cytometry, Fluorescence

Journal: PloS one

Article Title: Phagocytosis is the main CR3-mediated function affected by the lupus-associated variant of CD11b in human myeloid cells.

doi: 10.1371/journal.pone.0057082

Figure Lengend Snippet: Figure 2. Phagocytosis of hiC3b-coated fluorescent beads. The uptake by macrophages (A), PMNs (B), monocytes (C) and DCs (D) was quantified by flow cytometry and the data are represented as percentage of phagocytosis. Data are expressed as mean+/2SEM. Filled columns 77R/R individuals, open columns 77R/H-77H/H individuals. Paired T test was applied and the p values are indicated. doi:10.1371/journal.pone.0057082.g002

Article Snippet: The cDNA of the 77R CD11b allele was obtained from OriGene Technologies (Rockville, MD, USA, SC315229, NM000632.2), whilst the lupus susceptible allele (77H) was obtained by site-directed mutagenesis by changing the G at position 230 to an A (R77H).

Techniques: Flow Cytometry

Journal: PloS one

Article Title: Phagocytosis is the main CR3-mediated function affected by the lupus-associated variant of CD11b in human myeloid cells.

doi: 10.1371/journal.pone.0057082

Figure Lengend Snippet: Figure 3. Rosetting assay. Percentage of rosettes formed by CFSE- labelled RBC-hiC3b with freshly isolated PMNs from donors carrying the susceptible allele (77H/H, open symbol) or the common allele (77R/R, closed symbol). One representative assay out of 3 independent experiments. doi:10.1371/journal.pone.0057082.g003

Article Snippet: The cDNA of the 77R CD11b allele was obtained from OriGene Technologies (Rockville, MD, USA, SC315229, NM000632.2), whilst the lupus susceptible allele (77H) was obtained by site-directed mutagenesis by changing the G at position 230 to an A (R77H).

Techniques: Isolation

Journal: PloS one

Article Title: Phagocytosis is the main CR3-mediated function affected by the lupus-associated variant of CD11b in human myeloid cells.

doi: 10.1371/journal.pone.0057082

Figure Lengend Snippet: Figure 4. In vitro and in vivo PMN migration. (A) Migration of neutrophil cell lines through transwells seeded with mouse endothelial cells. PMNs migrated into the bottom chamber in response to MIP-2 were counted at different time points as indicated. Pooled results from at least 4 independent experiments are presented as mean 6 SEM. Itgam2/2 and wild type C57BL/6 neutrophil cell lines were used as controls. CD11b- deficient PMNs, known to have weaker endothelial interactions, migrated faster than the C57BL/6 and the hCD11b expressing cell lines (p,0.001 at 60 mns and p,0.05 at 90 mns). Statistical analysis by Bonferroni’s multiple comparison test. (B) Time course of the migration of freshly isolated human 77R/R or 77R/H neutrophils through a HUVEC layer in response to MIP-2. Pooled results from at least 4 independent experiments are presented as mean 6 SEM. (C, D) In vivo peritoneal migration of hCD11b-77R and hCD11b-77H PMN cells lines following i.p. injection of MIP-2 (C) and thioglycollate (D). The two hCD11b expressing PMN lines were labelled with DDAO or CFSE and adoptive transferred at a 1:1 ratio into C57BL/6 mice. Absolute numbers of labelled PMNs recovered from the peritoneum are shown. Data of one out of at least 3 independent experiments are presented. Bars indicate means. doi:10.1371/journal.pone.0057082.g004

Article Snippet: The cDNA of the 77R CD11b allele was obtained from OriGene Technologies (Rockville, MD, USA, SC315229, NM000632.2), whilst the lupus susceptible allele (77H) was obtained by site-directed mutagenesis by changing the G at position 230 to an A (R77H).

Techniques: In Vitro, In Vivo, Migration, Expressing, Comparison, Isolation, Injection

Journal: PloS one

Article Title: Phagocytosis is the main CR3-mediated function affected by the lupus-associated variant of CD11b in human myeloid cells.

doi: 10.1371/journal.pone.0057082

Figure Lengend Snippet: Figure 5. Cytokine response. Monocytes (A), DCs (B) were stimulated with 2 mg/ml and 10 mg/ml of TLR7/8 ligand (R848) respectively for 24 h. Cytokines quantified using a bead multiplex assay. Closed symbols: 77R/R cells, open symbols: 77R/H-77H/H cells. Each dot represents a single individual, bars denote means. No significant differences between the two CD11b genotypes. Statistical analysis by paired t test. (C, D) Modulation of TLR7/8-induced cytokine release by hiC3b-coated beads. Monocytes (C), DCs (D) were fed with hiC3b-coated beads one hour prior to R848 stimulation. The cytokine changes between the samples with and without CR3 pre-engagement with iC3b are shown with the p values indicated. Data are expressed as mean+/2SEM. The cytokine responses of 77R/R cells (black column) and 77R/H-77H/H cells (white columns) were not statistically different in paired assays. IL, interleukin; TNF-a, tumour necrosis factor alpha; IP-10, Interferon gamma-induced protein 10. doi:10.1371/journal.pone.0057082.g005

Article Snippet: The cDNA of the 77R CD11b allele was obtained from OriGene Technologies (Rockville, MD, USA, SC315229, NM000632.2), whilst the lupus susceptible allele (77H) was obtained by site-directed mutagenesis by changing the G at position 230 to an A (R77H).

Techniques: Multiplex Assay

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