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: 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: Journal of Translational Medicine

Article Title: A CD19/Fc fusion protein for detection of anti-CD19 chimeric antigen receptors

doi: 10.1186/1479-5876-11-23

Figure Lengend Snippet: Construction and characterization of the CD19-IgG 1 Fc fusion proteins. A . Diagrams of the lentiviral vector provirus constructs used to transduce the CD19sIg fusion genes. B . Reduced protein electrophoresis by SDS PAGE of fusion protein products after concentration and purification using Protein A Dynabeads. Centricon concentrated supernatant samples from fresh Pro293a™-CDM (A) , 293 T cells (B) , 293 T cells expressing CD19sIg1-3 (C) , and 293 T cells expressing CD19sIg1-4 (D) . Lanes E and F are the DYNAL purified extracts for CD19sIg1-3 and CD19sIg1-4 (respectively). Arrows indicate the predicted size of the monomer, 47 kDa for CD19sIg1-3 and 57 kDa for CD19sIg1-4. All samples were reduced prior to loading. C . Native protein electrophoresis of CD19sIg1-4 after concentration and purification using Protein A Dynabeads; arrow points to 171 kDa band (expected size for trimers of the fusion protein). D . Results of a comparative ELISA using FMC63 monoclonal capture antibody, of purified fusion proteins, in native (N) or denatured (D) forms. E . Results of ELISA using antibodies targeting human CD19 molecule (FMC63, HIB19, F-3 and 2E2B6B10), human CD20 (B9E9) and PSMA (YPSMA-1).

Article Snippet: The CD19-IgG 1 Fc fusion proteins, CD19sIg1-3 and CD19sIg1-4, were constructed by fusing either exons 1 to 3 (E13) or exons 1 to 4 (E14) of the human CD19 cDNA (Origene, Rockville, MD) to a human IgG 1 Fc (Fc) fragment [ ] by PCR-based cloning.

Techniques: Plasmid Preparation, Construct, Transduction, Protein Electrophoresis, SDS Page, Concentration Assay, Purification, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Journal of Translational Medicine

Article Title: A CD19/Fc fusion protein for detection of anti-CD19 chimeric antigen receptors

doi: 10.1186/1479-5876-11-23

Figure Lengend Snippet: Evaluation of CD19sIg1-4 fusion protein on primary human cell populations. Flow cytometry plots using FITC-conjugated anti-IgG Fc F(ab’) 2 fragment (FITC-anti-IgG Fc, left panels) or Alexa Fluor 488-labeled CD19sIg1-4 (AF488-CD19sIg1-4, right panels) for detection of 5% anti-CD19 CAR-transduced human primary T-cells mixed with (A) human peripheral blood mononuclear cells (PBMC), (B) NSG bone marrow (NSG BM), and (C) humanized NSG bone marrow ( hu NSG BM).

Article Snippet: The CD19-IgG 1 Fc fusion proteins, CD19sIg1-3 and CD19sIg1-4, were constructed by fusing either exons 1 to 3 (E13) or exons 1 to 4 (E14) of the human CD19 cDNA (Origene, Rockville, MD) to a human IgG 1 Fc (Fc) fragment [ ] by PCR-based cloning.

Techniques: Flow Cytometry, Labeling

Journal: Journal of Translational Medicine

Article Title: A CD19/Fc fusion protein for detection of anti-CD19 chimeric antigen receptors

doi: 10.1186/1479-5876-11-23

Figure Lengend Snippet: Evaluation of CD19sIg1-4 fusion protein for detection of anti-CD19 CAR-modified primary human T-cells. Flow cytometry plots demonstrating the sensitivity of detection of anti-CD19 CAR-transduced human primary T-cells mixed in increasing numbers of non-transduced (NT) T-cells using FITC-conjugated anti-IgG Fc F(ab’) 2 fragment (FITC-anti-IgG Fc, upper panels) or Alexa Fluor 488-labeled CD19sIg1-4 (AF488-CD19sIg1-4, lower panels).

Article Snippet: The CD19-IgG 1 Fc fusion proteins, CD19sIg1-3 and CD19sIg1-4, were constructed by fusing either exons 1 to 3 (E13) or exons 1 to 4 (E14) of the human CD19 cDNA (Origene, Rockville, MD) to a human IgG 1 Fc (Fc) fragment [ ] by PCR-based cloning.

Techniques: Modification, Flow Cytometry, Labeling

Journal: Journal of Translational Medicine

Article Title: A CD19/Fc fusion protein for detection of anti-CD19 chimeric antigen receptors

doi: 10.1186/1479-5876-11-23

Figure Lengend Snippet: Comparison of CD19sIg1-4 fusion protein to similar commercially available reagents. Flow cytometry plots of staining of primary human T-cells, non-transduced and 5% CAR-transduced, using FITC-conjugated F(ab’) 2 fragment goat anti-human IgG1 Fc γ (A) , biotinylated Protein L (B) , Alexa Fluor 488-labeled CD19sIg1-4 (C) and Alexa Fluor 488-labeled rhCD19-Fc fusion protein (D) . E . Results of staining of the same cell population after pre-incubation of Alexa Fluor 488-labeled CD19sIg1-4 with anti-CD19 monoclonal antibody FMC63.

Article Snippet: The CD19-IgG 1 Fc fusion proteins, CD19sIg1-3 and CD19sIg1-4, were constructed by fusing either exons 1 to 3 (E13) or exons 1 to 4 (E14) of the human CD19 cDNA (Origene, Rockville, MD) to a human IgG 1 Fc (Fc) fragment [ ] by PCR-based cloning.

Techniques: Comparison, Flow Cytometry, Staining, Labeling, Incubation

Journal: medRxiv

Article Title: In severe COVID-19, SARS-CoV-2 induces a chronic, TGF-β-dominated adaptive immune response

doi: 10.1101/2020.09.04.20188169

Figure Lengend Snippet: Peripheral blood activated B cells (CD27 high CD38 high ) were isolated and sorted by FACS for single cell sequencing. a) Percentage of CD27 high CD38 high cells among live B cells. Each sample is represented by a circle. The line indicates the median. Unpaired two sided Mann-Whitney U test. b) UMAP representation of 72277 CD27 high CD38 high sorted B cells from 9 COVID-19 ICU patients and 3 healthy controls. Between 2416 and 11229 cells were recovered per sample. Transcriptionally similar clusters were identified using shared nearest neighbor (SNN) modularity optimization (left). The combination of CD19, MS4A1, CD27, CD38, IFIT1, MIKI67, IRF4 and PRDM1 expression were used for annotation of the different activation/differentiation stages (right). c) Heatmap of genes that resemble markers for the six different clusters. Depicted are genes with a p-value < 0.01 (Wilcoxon rank sum test) after bonferroni correction, and an average absolute fold-change > log2(1.3). Shown are z-scores of the average expression. d) UMAP representation of the expression levels of selected signature genes for activated/differentiated B cells. e) UMAP representation of analyzed cells from one healthy control and two ICU patients, representing early (first week, patient #1) and late (>7days, patient #5) phase after ICU admission. f) Percentage of cells belonging to a defined cluster among all sequenced B cells per donor. Each dot represents one time point from one single donor. Donors were grouped as “HC” for healthy controls (n = 3), “1st week” for patients within 7 days after ICU admission (n = 6) and “Late” for patients who have been admitted to the ICU for more than a week at the time of analysis (n = 8). The line indicates the median. Significance was determined by using a two-sided analysis of varience (ANOVA) folowed by Tukey’s multiple comparison test with corrected p values as indicated in the figure.

Article Snippet: B cells were enriched from peripheral blood using StraightFrom® Whole Blood CD19 MicroBeads (Miltenyi Biotec) according to manufacturer’s instructions.

Techniques: Isolation, Sequencing, MANN-WHITNEY, Expressing, Activation Assay, Control, Comparison

Journal: medRxiv

Article Title: In severe COVID-19, SARS-CoV-2 induces a chronic, TGF-β-dominated adaptive immune response

doi: 10.1101/2020.09.04.20188169

Figure Lengend Snippet: a) 5-marker MELC panel of SARS-CoV2-positive and control lungs (SARS-CoV2-negative). Respective patient characteristics are given in supplementary Fig. 4a. Each image of each patient depicts the same field of view of the same section, sequentially stained with the fluorescence-labelled antibodies indicated and the nuclear stain DAPI. Magenta arrows indicate IgA2+IgA+CD27 + CD38 + cells (containing a nucleus). Images contain 2048 × 2048 pixels and are generated using an inverted wide-field fluorescence microscope with a 20x objective, a lateral resolution of 325 nm and an axial resolution above 5 µm. Scale bar: 100 µm. b) Absolute numbers of IgA2+IgA+CD27 + CD38 + cells per field of view in all MELC runs acquired (two runs per patient except for COVID-19_A with four runs and Control_B with one single run; see and supplementary Fig. 4b). Each field of view is represented by a circle. The line indicates the median. Unpaired two sided Mann-Whitney U test. c) Region of interest of one exemplary control and COVID-19 lung (as in a)), showing an overlay of the indicated markers. White arrows point out IgA2+CD27 + CD38 + cells. Scale bar: 20 µm. (d-e) Bronchoalveolar lavage (BAL) cells for single cell sequencing were enriched for CD45 + cells via MACS and live cells were further sorted using FACS (see supplementary Fig. 4c). d) UMAP of 5459 cells representing clusters containing T cells, B cells and CD14 -expressing cells from patient #1 on day 59 following ICU admission. B and T cell cluster identification based on BCR/TCR, CD19, CD3E, CD4 and CD8A expression (see supplementary Fig. 4d). UMAP representation of expression of TGFB1, IL21, CD40LG and IFNG . e) UMAP coordinates and clustering was computed for 433 and 2723 cells from patient #9 on days 31 and 46 following ICU admission, respectively. UMAP representation of expression of TGFB1, IL21, CD40LG and IFNG at the two different time points is shown side by side.

Article Snippet: B cells were enriched from peripheral blood using StraightFrom® Whole Blood CD19 MicroBeads (Miltenyi Biotec) according to manufacturer’s instructions.

Techniques: Marker, Control, Staining, Fluorescence, Generated, Microscopy, MANN-WHITNEY, Sequencing, Expressing