Journal: STAR Protocols

Article Title: Protocol for correlation analysis of the murine gut microbiome and meta-metabolome using 16S rDNA sequencing and UPLC-MS

doi: 10.1016/j.xpro.2022.101494

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Article Snippet: Remove CD3 + T cells by magnetic bead sorting (CD3 Selection Kit, Miltenyi Biotec). c. Purify splenic T cells by negative sorting using magnetic microbeads from the donor’s spleen (Pan T-Cell Isolation Kit, Miltenyi Biotec). d. Deliver 5×10 6 T-cell-depleted bone marrow (TCD-BM) cells with or without 5×10 5 splenic T cells to recipients on day 0 after lethal irradiation, while T cells are used as a source for aGVHD induction. e. Assess the animals for weight loss, clinical condition and disease progression twice a week according to the criteria of the disease model.

Techniques: Cell Isolation, Isolation, Control, Modification, Amplification, Sequencing, Software, Recombinant, Mass Spectrometry

Journal: Cell

Article Title: Memory B cell repertoire for recognition of evolving SARS-CoV-2 spike

doi: 10.1016/j.cell.2021.07.025

Figure Lengend Snippet: SARS-CoV-2 spike-specific mAb binding profiles (A) Cells recovered from two sorting strategies, shown in dot plots as percentages of total CD19 + cells. Left: IgG + CD27 + cells from 18 donors and the subset of those that expressed S-binding BCRs. Right: cells from three donors expressing S-binding BCRs and sorted to recover principally those that did not bind RBD. (B) Summary of all productive mAbs (recombinant human IgG1) screened by ELISA (with recombinant S ectodomain trimer) and cell-surface expression assays (both 293T and yeast cells). Total numbers in the center of each of pie chart; numbers and color codes for the indicated populations shown next to each chart. To the right of the charts for the two sorting strategies are bar graphs showing frequencies of SARS-CoV-2 RBD and NTD binding mAbs for those subjects from whom at least ten paired-chain BCR sequences were recovered. (C) Binding to a panel of S proteins and SARS-CoV-2 subdomains, listed on the left, as determined by both ELISA and by association with S expressed on the surface of 293T cells or with RBD or NTD expressed on the surface of yeast cells, for S + sorted (left) and S + RBD − sorted cells (right). Left panel, 157 clones bound to S and an additional one bound to only RBD but not S. Pink indicates ELISA screens. Blue indicates cell-based screens. Each short section of a row represents an antibody. The rows labeled VH mutation and VL mutation are heatmaps of counts (excluding CDR3) from alignment by IgBLAST, with the scale indicated. (D) Dot plots of VH and VL mutation counts in mAbs that bound RBD, NTD, S2, and a “broad CoV group” that included MERS, HKU1, and OC43. ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; nonparametric Kruskal-Wallis multiple comparison. Horizontal lines show mean ± SEM. See also Figure S1 .

Article Snippet: Human CD19 MicroBeads , Miltenyi Biotec , Cat# 130-050-301, RRID: AB_2848166.

Techniques: Binding Assay, Expressing, Recombinant, Enzyme-linked Immunosorbent Assay, Clone Assay, Labeling, Mutagenesis, Comparison

Journal: Cell

Article Title: Memory B cell repertoire for recognition of evolving SARS-CoV-2 spike

doi: 10.1016/j.cell.2021.07.025

Figure Lengend Snippet: Sorting strategy for SARS-CoV-2-specific memory B cells and characterization of monoclonal antibodies, related to Figure 1 (A) Representative flow cytometry plots showing CD19 + , CD27 + , SARS-CoV-2 S-binding B cells from a convalescent subject (C12, top row) and a pre-pandemic control (bottom row). PBMCs were pre-enriched with CD19 magnetic beads then gated on live IgD − IgM-IgG + CD27 + and finally on S (B) Representative flow cytometry plots showing S-positive, RBD-negative B cells for three convalescent subjects and a pre-pandemic control, sorted as in (A) except for the S gate. (C) Representative flow plot of mAb supernatant bound to SARS-CoV-2 S on HEK293T cells. Cells were gated on DAPI − GFP + population. (D) Representative flow plot of mAb supernatant bound to SARS-CoV-2 RBD on yeast. cMyc tag indicated yeast that expressed RBD. (E) Representative flow plot of mAb supernatant bound to SARS-CoV-2 NTD on yeast. cMyc tag indicated yeast that expressed NTD. See Figure 1 C for the screening color scheme. (F) Bar graph of Log 10 (EC 50 ) of antibodies targeting RBD, NTD and S2 using ELISA and cell-based assay. EC 50 (μg/mL), RBD (n = 23), NTD clusters (n = 15) and S2 (n = 15). ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; Paired nonparametric Wilcoxon test. Data are mean values ± SEM (G) Dot plot of Log 10 (EC 50 ) of antibodies in the indicated bins by cell-based assay. Antibodies are from subjects G32 and C41, sorted with S. Each dot represents one monoclonal antibody. EC 50 (μg/mL), 13-39 days (n = 13), 40-63 days (n = 8). No significance; nonparametric Mann-Whitney test. Data are mean values ± SEM.

Article Snippet: Human CD19 MicroBeads , Miltenyi Biotec , Cat# 130-050-301, RRID: AB_2848166.

Techniques: Bioprocessing, Flow Cytometry, Binding Assay, Control, Magnetic Beads, Enzyme-linked Immunosorbent Assay, Cell Based Assay, MANN-WHITNEY

Journal: Cell

Article Title: Memory B cell repertoire for recognition of evolving SARS-CoV-2 spike

doi: 10.1016/j.cell.2021.07.025

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Article Snippet: Human CD19 MicroBeads , Miltenyi Biotec , Cat# 130-050-301, RRID: AB_2848166.

Techniques: Virus, Recombinant, Enzyme-linked Immunosorbent Assay, Saline, Random Hexamer, Reverse Transcription, Transfection, Luciferase, Plasmid Preparation, Flow Cytometry, Software

Journal: Annals of Translational Medicine

Article Title: KIAA0101 knockdown inhibits cell proliferation and induces cell cycle arrest and cell apoptosis in chronic lymphocytic leukemia cells

doi: 10.21037/atm-21-626

Figure Lengend Snippet: KIAA0101 expression was markedly up-regulated in CD19+ B cells isolated from the peripheral blood of CLL patients. The mRNA expression level of KIAA0101 was examined through RT-qPCR in CD19+ B cells isolated from the peripheral blood of 6 healthy people and 26 CLL patients. *P<0.05. CLL, chronic lymphocytic leukemia; RT-qPCR, real-time quantitative PCR.

Article Snippet: CD19 + B cells were freshly isolated from peripheral blood mononuclear cells (PBMCs) through magnetic cell sorting (MACS) of Lymphoprep-separated buffy coats based on MACS CD19 Microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) (>98%).

Techniques: Expressing, Isolation, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

Journal:

Article Title: Gab3 -Deficient Mice Exhibit Normal Development and Hematopoiesis and Are Immunocompetent

doi: 10.1128/MCB.23.7.2415-2424.2003

Figure Lengend Snippet: Expression of Gab3 protein. (A) Gab3 was immunoprecipitated using polyclonal anti-Gab3 serum from protein lysates of the indicated tissues of mice and detected by Western blot analysis using the Gab3-specific monoclonal antibody P5G9. (B) Primary BM cells isolated from mice were differentiated into macrophages by culturing them for 6 days in M-CSF. IP and Western blot analysis was done as described in the legend for panel A by using normalized protein lysates of cells before and after M-CSF treatment. (C) A single-cell suspension of C57BL/6 BM was separated by magnetic bead-conjugated antibodies and AutoMACS into CD19+ B lymphocytes and CD19− cells. The cell suspension of spleen cells was separated into CD4+ T-cell, CD8+ T-cell, and CD19+ B-cell subpopulations by AutoMACS. Thymocytes were separated into CD4+ CD8−, CD4− CD8+, and CD4+ CD8+ subpopulations by flow cytometry. Expression of Gab3 in all these cell populations was analyzed by IP and Western blot analysis as described above, using Gab3-specific antibodies.

Article Snippet: CD19 + B cells were positively selected from single-cell suspensions of Gab3 −/− and littermate control BM by labeling the cells with magnetic bead-conjugated antibodies against CD19 (Miltenyi Biotec, Auburn, Calif.), followed by AutoMACS magnetic bead sorting according to the manufacturer's instructions.

Techniques: Expressing, Immunoprecipitation, Western Blot, Isolation, Suspension, Flow Cytometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Differential influence on regulatory B cells by T H 2 cytokines affects protection in allergic airway disease.

doi: 10.4049/jimmunol.1800206

Figure Lengend Snippet: Mice were sensitized to OVA in Alum twice by intraperitoneal injection. Purified splenic B cells from BALB/c mice were cultured with IL-5/mCD40L-Fb for five days then FACS sorted into CD5+ and CD5− subsets. Sensitized mice received i.v. adoptive transfers of 2×106 sorted B cells/mouse prior to intratracheal challenge daily with 10μg OVA for 3 days. (A) Airway hyperresponsiveness was assessed by plethysmography. (B) BAL fluid was collected and assessed by ELISA for IL-4. (C and D) Cells in the BAL fluid were counted and assessed by flow cytometry for eosinophils (CD45+CD4−CD19−Ly6G−SiglecF+MHCII−). (E and F) Regulatory T cells in the lung and mediastinal lymph nodes (CD45+CD3+CD4+CD25+FoxP3+) were determined by intracellular flow cytometry. Data are a single representative experiment of 3 independent experiments (mean ± SEM); 3–5 mice/group. Statistics are from Mann Whitney test comparing treatment groups to allergic control. P values are indicated by: * P<0.05.

Article Snippet: Purified mouse B cells were obtained from single cell suspensions derived from the spleens of naive 8–12 week old mice by anti-mouse CD19 MACS magnetic bead positive selection (Miltenyi Biotec, Auburn, CA) using the manufacturer protocol.

Techniques: Injection, Purification, Cell Culture, Enzyme-linked Immunosorbent Assay, Flow Cytometry, MANN-WHITNEY, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Differential influence on regulatory B cells by T H 2 cytokines affects protection in allergic airway disease.

doi: 10.4049/jimmunol.1800206

Figure Lengend Snippet: OVA-sensitized mice received 2×106 sorted CD5+ B cells/mouse obtained from IL-5/mCD40L-Fb-stimulated cultures of splenic B cells from wild-type BALB/c or IL-10−/− mice. Transfer recipients and untreated controls were then challenged daily with 10μg OVA for 3 days. (A) Airway hyperresponsiveness was assessed by plethysmography. (B) BAL fluid was collected and assessed by ELISA for IL-4. (C and D) Cells in the BAL fluid were counted and assessed by flow cytometry for eosinophils (CD45+CD4−CD19−Ly6G−SiglecF+MHCII−). Data is from one experiment representative of 3 independent experiments (mean ± SEM) with at least 6 mice/group. Statistics were done using Mann Whitney tests comparing each treatment group to allergic control. P values are indicated by: * P<0.05, **P<0.01, or exact values.

Article Snippet: Purified mouse B cells were obtained from single cell suspensions derived from the spleens of naive 8–12 week old mice by anti-mouse CD19 MACS magnetic bead positive selection (Miltenyi Biotec, Auburn, CA) using the manufacturer protocol.

Techniques: Enzyme-linked Immunosorbent Assay, Flow Cytometry, MANN-WHITNEY, Control

Journal: Oncology Reports

Article Title: Constitutive activation of NF-κB signaling by NOTCH1 mutations in chronic lymphocytic leukemia

doi: 10.3892/or.2015.3762

Figure Lengend Snippet: High expression of ICN proteins in the cytoplasm and nuclei of NOTCH1-mutated CLL cells, detected by immunofluorescence (x400). The images are representative of three (A) mutated and (B) five unmutated CLL cells. (C) In CLL cells carrying NOTCH1 mutations, the ICN protein was abundantly expressed in the cytoplasm and nuclei, at significantly higher levels than that observed in unmutated and normal CD19 + B cells ( * P<0.01).

Article Snippet: The human CD19 + magnetic bead sorting kit was obtained from Miltenyi Biotec (Shanghai, China).

Techniques: Expressing, Immunofluorescence