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: Inducible immortalized Dendritic Cells enable antigen-specific antibody production in a murine in vitro Immunization model

doi: 10.1371/journal.pone.0339883

Figure Lengend Snippet: (A) Microscopic images from immature BM-DCs isolated from a CB57Bl/6 mouse (left panel), iniDCs after 7 days stimulation with Dex/Dox (middle panel), and de-iniDCs after 3 days of deinduction (right panel). Upon deinduction, de-iniDCs display an adherent phenotype as well as typical dendritic extensions indicated by arrows. During their immortal period, iniDCs lose typical dendritic morphology, become round-shaped, and pass over in suspension. (10 x magnification, bar = 50 µm) (B) Respectively, 5 x 10 5 BM-DCs, iniDCs, and de-iniDCs were stained with fluorochrome-conjugated antibodies against specific dendritic surface markers CD11c, MHC II, CD40, and CD86 and analyzed by flow cytometry. Dead cells were removed by PO-PRO TM -1 Iodide staining. Experiments were performed 3 times independently. Given is the result of one representative experiment. (C) Displayed are the geometric means (MFI) of the cell surface markers, with the background signal subtracted. dark grey = BM-DCs, white = iniDCs, light grey = de-iniDCs, (n = 6), two-tailed t-test (p < 0,05 = *; p < 0,01 = **; p < 0,001 = ***).

Article Snippet: Before analysis, iniDCs and de-iniDCs were additionally stained with PE-conjugated recombinant human-anti-mouse-CD11c-IgG antibody (Miltenyi Biotec, Bergisch Gladbach, Germany, Ref.: 130-110-701, Lot: 5190521242) and 1 μg/ml PO-PRO TM -1 Iodide (Thermo Fisher, Massachusetts, USA) as described above.

Techniques: Isolation, Suspension, Staining, Flow Cytometry, Two Tailed Test

Journal: PLOS One

Article Title: Inducible immortalized Dendritic Cells enable antigen-specific antibody production in a murine in vitro Immunization model

doi: 10.1371/journal.pone.0339883

Figure Lengend Snippet: (A) Respectively, 5 x 10 5 BM-DCs, iniDCs, and de-iniDCs were stained for CD11c and PO-PRO-1 Iodide to define the amount of living CD11c + cells by flow cytometry. After 9 days only 67% (±3.1%) of BM-DCs were alive, 7 days after Dex/Dox stimulation iniDCs displayed 83% (± 2.5%) living cells, and upon deinduction of 3 days vitality decreased to 70% (± 3.8%) of de-iniDCs, n = 3 (B) Vitality of iniDCs was stable for 42 days. After 21 days, 88% (± 2.5%, n = 3) and after 42 days, 87% of the CD11c + cells were alive. (C) For proliferation analysis best results were achieved by staining 35 x 10 5 iniDCs with 10 µM CFSE in PBS only. CD11c + cells were analyzed at day 1 (black) and 11 (green) using the BD FACSDAria III TM as well as an unstained control (grey). (D) de-iniDCs were stained with CFSE and measured at day 0, 1, 2, 3 and 4 as well as an unstained control.

Article Snippet: Before analysis, iniDCs and de-iniDCs were additionally stained with PE-conjugated recombinant human-anti-mouse-CD11c-IgG antibody (Miltenyi Biotec, Bergisch Gladbach, Germany, Ref.: 130-110-701, Lot: 5190521242) and 1 μg/ml PO-PRO TM -1 Iodide (Thermo Fisher, Massachusetts, USA) as described above.

Techniques: Staining, Flow Cytometry, Control

Journal: PLOS One

Article Title: Inducible immortalized Dendritic Cells enable antigen-specific antibody production in a murine in vitro Immunization model

doi: 10.1371/journal.pone.0339883

Figure Lengend Snippet: (A) 5 x 10 5 de-iniDCs were stimulated with or without 2 µg/ml DQ-Ova for 60 min at 37°C and measured by fluorescence microscopy using the BZ-800 Biozero (10 x magnification, bar = 50 µm), (B) 1 x 10 5 C57Bl/6 BM-DCs and de-iniDCs were challenged with 1 µg/ml DQ-Ova for 60 min at 37°C (black) and analyzed by flow cytometry using the Attune ® Acoustic Focusing Cytometer. Controls are depicted in grey. Given is the result of one representative experiment out of three. (C) After maturation of the de-iniDCs with VP1, respectively 5 x 10 5 cells were stained with fluorochrome-labeled antibodies against maturation markers CD11c, MHC II, CD40, or CD86. To exclude dead cells 1 µg/ml PO-PRO-1 TM Iodide was added before analysis using the Attune® Acoustic Focusing Cytometer. Unstained control is in light grey, immature DCs in dark grey, and mature DCs are in black. Given is one representative result out of three. (D) Displayed are the geometric means (MFI) of the different surface receptors before and after maturation with VP1. The background signal was subtracted. (n = 8), two-tailed t-test (p < 0,05 = *; p < 0,01 = **; p < 0,001 = ***) (E) 1x10 6 de-iniDCs were matured with 15 µg/ml VP1 for 24 h, and supernatant was analyzed for IL-10 and IL-12p70 by ELISA. (n = 3).

Article Snippet: Before analysis, iniDCs and de-iniDCs were additionally stained with PE-conjugated recombinant human-anti-mouse-CD11c-IgG antibody (Miltenyi Biotec, Bergisch Gladbach, Germany, Ref.: 130-110-701, Lot: 5190521242) and 1 μg/ml PO-PRO TM -1 Iodide (Thermo Fisher, Massachusetts, USA) as described above.

Techniques: Fluorescence, Microscopy, Flow Cytometry, Cytometry, Staining, Labeling, Control, Two Tailed Test, Enzyme-linked Immunosorbent Assay