Journal: American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons

Article Title: Porcine antigen presenting cells produce soluble adjuvants that stimulate B cells within and across the species.

doi: 10.1034/j.1600-6143.2003.00091.x

Figure Lengend Snippet: Figure 4: Porcine antigen presenting cells (pAPCs) induce proliferation of human B cells: Human peripheral blood lymphocytes were separated into CD19+ve (B-cell enriched) and CD19–ve (B-cell depleted) fractions by magnetic bead separation, then labeled with CFSE and cocultured for 96 h with no addition, 1 : 40 pGM-CSF APCs (GM-pAPCs), or 1 : 40 pGM-CSF + pIL-4 APCs (GM-IL-4-APCs). Cells were surface stained for CD19 (CD19-PE) and proliferation was measured by flow cytometry on the basis of CFSE dilution. Examples of analysis of B-cell enriched and B-cell depleted populations (following PI exclusion) are shown for no addition and pAPC coculture. B-cell enriched fractions (upper panels) were predominantly CD19+ve and demonstrated CFSE dilution only in the presence of pAPCs. B-cell depleted fractions (lower panels) were predominantly CD19–ve and demonstrated negligible cell division under all conditions. When results were expressed graphically (lower figure) as mean percent divided ± SD of triplicate samples for each condition, there were significant rates of cell division in the B-cell enriched but not the B-cell- depleted populations compared with control conditions. yp < 0.05 for values greater than the value with no addition.

Article Snippet: Purification of B cells was carried out by magnetic separation using antihuman CD19 microbeads and a magnetic separation column according to the manufacturer’s instructions (Miltenyi Biotech Inc., Auburn, CA).

Techniques: Labeling, Staining, Flow Cytometry, Control

Journal: American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons

Article Title: Porcine antigen presenting cells produce soluble adjuvants that stimulate B cells within and across the species.

doi: 10.1034/j.1600-6143.2003.00091.x

Figure Lengend Snippet: Figure 5: Porcine antigen presenting cells (pAPCs) induce proliferation of murine B cells: Murine whole lymph node cells from lipopolysaccharide-responsive (C3H/HeN) and LPS-unresponsive (C3H/HeJ) strains were labeled with CFSE and cocultured for 72 h with irradiated pAPCs generated using pGM-CSF alone (pGM-CSF APCs) or pGM-CSF combined with pIL-4 (pGM-CSF/ pIL-4 APCs) at ratios of 40 : 1. Division of B-cell and T-cell subsets was detected by three-color flow cytometric analysis using PI exclusion, surface staining for CD19 (B cells) or Thy 1.2 (T cells), and CFSE fluorescence dilution. Examples of PI-gated dot-plots and CFSE fluorescence histograms gated on B cells (aCD19-PE: left upper panels) or T cells (aThy 1.2-PE: left lower panels) are shown for C3H/HeN cells cultured with No APCs, pGM-CSF APCs or pGM-CSF/pIL-4 APCs. Results are shown graphically for B cells (CD19+ve/PI–ve: upper graph) and T cells (Thy 1.2+ve/PI–ve: lower graph) and are expressed as the mean– SD percentage of viable CD19 or Thy1.2-positive cells that had undergone division on the basis of reduced CFSE fluorescence. Co-culture of lymph node cells from both murine strains was associated with significantly increased rates of B-cell division compared with control conditions (no APCs). pGM-CSF/pIL-4 APCs induced significantly greater B-cell proliferation compared with pGM-CSF APCs. Rates of B-cell division for APC-stimulated cultures were between 65% and 80%. Significant increases in T-cell division compared with control conditions also occurred for C3N/HeN and C3N/HeJ strains using both types of pAPC but were of low magnitude (between 20 and 35%). Stimulation of lymph node cells from which B cells had been depleted (T-cell enriched: inset graph) did not alter the magnitude of the T-cell response to pAPC stimulation. Basal B-cell and T-cell proliferation was greater for C3H/HeJ compared with C3H/HeN lymph node cells but results were qualitatively similar for both strains. *p< 0.05 compared with control conditions (No APCs), yp < 0.05 for pGM-CSF/pIL-4 APCs compared with pGM-CSF APCs.

Article Snippet: Purification of B cells was carried out by magnetic separation using antihuman CD19 microbeads and a magnetic separation column according to the manufacturer’s instructions (Miltenyi Biotech Inc., Auburn, CA).

Techniques: Labeling, Irradiation, Generated, Staining, Fluorescence, Cell Culture, Co-Culture Assay, Control

Journal: American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons

Article Title: Porcine antigen presenting cells produce soluble adjuvants that stimulate B cells within and across the species.

doi: 10.1034/j.1600-6143.2003.00091.x

Figure Lengend Snippet: Figure 6: Porcine antigen presenting cells (pAPCs) migrate to lymph nodes and induce B-cell expansion in vivo: (A) Flow cytometric analysis of cells from left and right inguinal lymph nodes 6 h following subcutaneous inoculation of 3 105 CFSE- labeled pGM-CSF APCs to the left inguinal region and PBS to the right inguinal region. Fluorescent cells are present in the node draining the left but not the right inguinal region. (B) Groups of five B6 mice were inoculated subcutaneously with 50 mL of PBS to the right inguinal region or with one of three doses of pGM-CSF APCs (2 105, 2 104, 2 103) to the left inguinal region. Five days later all nodes were individually dissected and total cells counted. Surface staining for CD19 and flow cytometry was used to determine the B-cell percentage for each node. Results (expressed as mean ± SD, PBS vs. pAPC inoculations) for each group are shown graphically for B-cell percentage (% CD19+ve, left graph) and for total B-cell numbers per node (right graph). A significant, dose-dependent increase in B-cell percentage and total B-cell numbers occurred in lymph nodes draining pAPC inoculation sites. yp < 0.05 for pAPC vs. PBS.

Article Snippet: Purification of B cells was carried out by magnetic separation using antihuman CD19 microbeads and a magnetic separation column according to the manufacturer’s instructions (Miltenyi Biotech Inc., Auburn, CA).

Techniques: In Vivo, Labeling, Staining, Flow Cytometry

Journal: Stem cells international

Article Title: Feasibility and Efficiency of Human Bone Marrow Stromal Cell Culture with Allogeneic Platelet Lysate-Supplementation for Cell Therapy against Stroke.

doi: 10.1155/2016/6104780

Figure Lengend Snippet: Figure 4: Flow cytometric analysis of the surface markers. Gray lines: each specific antibody (CD44, CD90, CD105, CD106, CD146, CD166, CD19, and CD45); black lines: each isotopic antibody.

Article Snippet: The hBMSCs cultured with PL in CPC were suspended with PBS containing 3% FCS.They were incubated with either a mouse monoclonal antibody against human CD19 (R&D Systems; dilution, 1 : 100), CD44 (R&D Systems; 1 : 100), CD45 (R&D Systems; 1 : 100), CD90 (R&D Systems; 1 : 100), CD105 (R&D Systems; 1 : 100), CD106 (R&D Systems; 1 : 100), CD146 (R&D Systems; 1 : 100), CD166 (R&D Systems; 1 : 100), or each mouse isotypic control for 30min on ice.

Techniques:

Journal: bioRxiv

Article Title: Inhibition of polo-like kinase 1 (PLK1) facilitates reactivation of gamma-herpesviruses in B-cell lymphomas and their elimination

doi: 10.1101/2020.10.08.330548

Figure Lengend Snippet: (A) Total RNAs were extracted from the fixed diffuse large B-cell lymphoma (DLBCL) tissue samples of HIV-negative (HIV-, n=3) and HIV-positive (HIV+, n=5) individuals. mRNA level of PLK1 in these tissue samples was measured by RT-PCR and normalized to GAPDH. (B) A PE-conjugated CD19 antibody was used for B cell enrichment from PBMCs of HIV-infected, aviremic patients (HIV+, n=5) or healthy donors (HIV-, n=5). Purity of isolated CD19 + B cells was confirmed by flow cytometry analysis. (C) mRNA level of PLK1 in above isolated B cells (B) was measured by RT-PCR and normalized to GAPDH. (D) Protein level of intracellular PLK1 in above isolated B cells (B) was measured by immunofluorescence. Percentage of PLK1-expressing cells was determined by flow cytometry analysis. (F) HIV-1 IIIB infected Jurkat cells were co-cultured with BJAB cells that were pre-stained with CFSE. Protein level of intracellular PLK1 in CFSE-labeled BJAB cells was measured by immunofluorescence. Percentage of PLK1-expressing cells was determined by flow cytometry analysis. (G-I) Protein level of PLK1 in BJAB (G), Atata-BX1 (H), or TREx BCBL1-Rta (I) cells treated with recombinant Nef (rNef) protein at the increasing dose or DMSO was measured by immunoblotting. GAPDH was used as a loading control. Results were calculated from n=3 independent experiments and presented as mean ± SD (* p<0.05; two-tailed paired Student t-test).

Article Snippet: Portion of isolated B cells was incubated with CD19 antibody (1/200 of stock, Milteny) for 30 mins to determine the purity using BD Accuri C6 Plus with corresponding optical filters.

Techniques: Reverse Transcription Polymerase Chain Reaction, Infection, Isolation, Flow Cytometry, Immunofluorescence, Expressing, Cell Culture, Staining, Labeling, Recombinant, Western Blot, Control, Two Tailed Test