Intestinal plasmablasts/plasma cells were isolated as previously described (Benckert et al., 2011 (link)). In brief, lamina mucosa and propria were dissected from lamina muscularis mucosae by blunt preparation, and 2–3-mm tissue pieces were digested using 0.1% DNase and 0.1% collagenase followed by discontinuous Ficoll density gradient centrifugation (GE Healthcare). Purified lamina propria lymphocytes were stained on ice with fluorochrome-coupled anti-human CD38 FITC, anti-human CD27 PE, anti-human CD19 PECy7, anti-human IgG APC (all from BD Bioscience) or anti-human IgA APC (Jackson Laboratory). Single CD38+CD27+IgA+ or CD38+CD27+IgG+ plasmablasts/plasma cells were sorted into 96-well PCR plates using a FACSVantage cell sorter with Diva configuration (BD Bioscience), snap frozen on dry ice, and stored at −80°C until further processing.
Anti human cd19 pecy7
Manufactured by BD
Sourced in United States
Anti-human CD19 PECy7 is a fluorescent-labeled antibody that binds to the CD19 antigen expressed on the surface of human B cells. It is used for the identification and enumeration of B cells in flow cytometry analysis.
Automatically generated - may contain errors
Lab products found in correlation
Anti human cd27 pe, by BD (2 mentions)
Anti human igg apc, by BD (1 mentions)
Ficoll density gradient centrifugation, by GE Healthcare (1 mentions)
Facsvantage cell sorter, by BD (1 mentions)
Annexin v 7aad apoptosis kit, by BD (1 mentions)
Anti human cd3 pacific blue, by BD (1 mentions)
Anti human cd15 apc, by Miltenyi Biotec (1 mentions)
Brefeldin a, by Merck Group (1 mentions)
Saponin, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Carl Roth (1 mentions)
Rpmi 1640 culture medium, by Thermo Fisher Scientific (1 mentions)
Anti human cd38 percp cy5, by BD (1 mentions)
Isotype matched control igg, by BD (1 mentions)
Anti human cxcr5 percp cy5, by BD (1 mentions)
Anti human cd3 bv510, by BD (1 mentions)
Ficoll paque plus, by Cytiva (1 mentions)
Facsaria 2, by BD (1 mentions)
Anti human cd3 apc, by BD (1 mentions)
Pe anti human cd86, by BioLegend (1 mentions)
Lsr fortessa cytometer, by BD (1 mentions)
4 protocols using anti human cd19 pecy7
1
Intestinal plasmablast/plasma cell isolation
2
Profiling Immune Cell Responses to Stimuli
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Lipopolysaccharide (LPS), lectin from Phaseolus vulgaris (PHA), Brefeldin A, Saponin, and carboxyfluorescein diacetate N-succinimidyl ester (CFSE) were from Sigma-Aldrich Co., St Louis, MO, USA. Paraformaldehyde was from Carl Roth GmbH + Co. KG (Karlsruhe, Germany). Recombinant human macrophage colony stimulating factor (rhM-CSF) was from ImmunoTools GmbH (Friesoythe, Germany).
For flow cytometry, Privigen® human immunoglobulin from CSL Bhering, (King of Prussia, PA, USA) was used. Anti-human-IL1β-PE, anti-human-CD3-Pacific-blue, anti-human-CD14-APC-Cy7, anti-human-CD19-PE-Cy7, and Annexin V/7AAD apoptosis kit were from BD Biosciences (San Jose, CA, USA) and anti-human-CD15-APC was from Miltenyi Biotech GmbH (Bergisch Gladbach, Germany).
For flow cytometry, Privigen® human immunoglobulin from CSL Bhering, (King of Prussia, PA, USA) was used. Anti-human-IL1β-PE, anti-human-CD3-Pacific-blue, anti-human-CD14-APC-Cy7, anti-human-CD19-PE-Cy7, and Annexin V/7AAD apoptosis kit were from BD Biosciences (San Jose, CA, USA) and anti-human-CD15-APC was from Miltenyi Biotech GmbH (Bergisch Gladbach, Germany).
3
Phenotyping of Peripheral Blood Lymphocytes
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Peripheral blood mononuclear cells (PBMCs) were isolated by density-gradient centrifugation using Ficoll-Paque Plus (Amersham Biosciences, Buckinghamshire, UK). The cells were then resuspended to 1 × 106 per mL in RPMI-1640 culture medium (Invitrogen, Carlsbad, CA, USA) containing 10% fetal calf serum, and stained with the following monoclonal antibodies: antihuman CD3-BV510, antihuman CD4-APC-H7, antihuman CXCR5-PerCP-Cy5.5, antihuman CXCR3-PE-Cy7, antihuman CCR6-PE, antihuman CD45RA-PE-CF594, antihuman ICOS-BV421, antihuman PD1-FITC, antihuman CD27-PE, antihuman CD20-APC-H7, antihuman CD38-PerCP-Cy5.5, antihuman CD19-PE-cy7 or isotype-matched control IgG (BD PharMingen San Diego, CA, U.S.). After washing twice with phosphate-buffered saline (containing 0.1% (w/v) NaN3), the samples were analyzed using FACS Aria II (BD Sciences, San Jose, USA); at least 50,000 events per sample were analyzed using FlowJo software (v7.6.2). The absolute count of cells was calculated on the basis of the cell proportion multiplied by the absolute number of lymphocytes.
4
Immunophenotyping of PBMCs Stimulated with OMVs
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Peripheral blood mononuclear cells were stimulated with either a 1, 10, or 25 μg/mL suspension of OMVs from both strains or a 5 μg phytohemagglutinin (PHA) solution for 12, 24, and 48 h. Plates were incubated at 37°C under 5% CO2. Monoclonal antibodies (mAbs) coupled to the following fluorochromes were used: anti-human CD91-eFluor 660 (eBioscience), anti-human CD3-APC (BD PharmingenTM), anti-human CD19-PE-Cy7 (BD Biosciences), anti-human PD-L1-PE (BioLegend), anti-human PD-1-FITC (BioLegend), anti-human CD86-PE (BioLegend), anti-human CD69-TRI-COLOR (Molecular Probes). After stimulation, 100 μL of supernatant were collected from wells and stored at −70°C until usage. Then, PBMCs were collected and washed with FACS buffer, followed by incubation with diluted mAbs in FACS buffer for 1 h at 4°C (protecting the reaction from light). Finally, cells were washed with FACS buffer, fixed with PBS-PFA (Paraformaldehyde) 1%, and resuspended in 400 μL of FACS buffer. Samples were analyzed in LSRFortessaTM cytometer (BD Biosciences), providing a total number of 30,000 events, and the resulting data was analyzed with the aid of the FlowJo® software.
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