After overnight cytokine induction, B and T cells were removed from the samples using the EasySepTM Human CD19/CD3 Positive Selection Kit II (STEMCELL Technologies, Vancouver, BC, Canada) according to manufacturer’s instructions with the difference that the supernatant was kept and the B and T cells containing tubes were discarded. Cells were counted and a sample of 1 × 105 cells was used for flow cytometry to check for successful depletion of the lymphocytes. In brief, cells were washed with PBS-EB (PBS with 2.5 mM EDTA and 1% BSA) and incubated with anti-human CD2-FITC and anti-human CD19-PE-Cy7 (both from Biolegend, San Diego, CA, USA) antibodies for 30 min at 4 °C in the dark. The stained cells were washed twice in PBS-EB and resuspended in PBS containing 1 μg/mL DAPI (Sigma-Aldrich, St. Louis, MO, USA). Flow cytometry was performed on the BD LSR II (Becton Dickinson, Franklin Lakes, NJ, USA) machine and the data analyzed using FlowJo software (FlowJo, LLC, Becton Dickinson, Franklin Lakes, NJ, USA).
Anti human cd19 pe cy7
Manufactured by BioLegend
Sourced in United States
Anti-human CD19-PE-Cy7 is a fluorescently-labeled monoclonal antibody that binds to the CD19 antigen expressed on human B cells. It can be used for the identification and enumeration of B cells in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti mouse cd45 percp cy5, by BioLegend (2 mentions)
Anti human cd11b pe, by BD (2 mentions)
Anti mouse cd16 cd32, by BioLegend (2 mentions)
Anti human cd34 pe, by BioLegend (2 mentions)
Rbc lysis buffer, by Santa Cruz Biotechnology (2 mentions)
Anti human cd3 pe dazzle 594, by BioLegend (2 mentions)
Anti mouse cd45 percp, by BD (2 mentions)
Anti human cd45 apc, by BD (2 mentions)
Anti human cd2 fitc, by BioLegend (1 mentions)
Lsr 2, by BD (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Human trustain fcx, by BioLegend (1 mentions)
Facs lysing solution, by BD (1 mentions)
Anti human cd8 apc h7, by BD (1 mentions)
Lsr fortessa 4, by BD (1 mentions)
Anti human cd38 apc, by BioLegend (1 mentions)
Anti human cd3 pacific blue, by BD (1 mentions)
Anti human cd15 apc, by Miltenyi Biotec (1 mentions)
Lectin from phaseolus vulgaris pha l, by Merck Group (1 mentions)
Anti human cd14 apc cy7, by BioLegend (1 mentions)
8 protocols using anti human cd19 pe cy7
1
Lymphocyte Depletion from Cell Samples
2
Immune Cell Phenotyping by Flow Cytometry
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Staining buffer was PBS supplemented with 0.5% FCS and 4 mM EDTA. Whole blood collected in Lithium Heparin vacutainers was lysed and fixed with BD FACS lysing solution (Becton Dickinson) and lymphocytes permeabilized with BD FACS permeabilising solution 2 (Becton Dickinson) according to the manufacturer’s instructions. Cells were then resuspended in 50 μl of staining buffer containing anti-human CD4-BV510 (Becton Dickinson, 1:200 dilution), anti-human CD8-APC-H7 (Becton Dickinson, 1:400 dilution), anti-human CD19-PE-Cy7 (Biolegend, 1:200 dilution), anti-human CD38-APC (Biolegend, 1:400 dilution), anti-human Perforin-PE (Biolegend, 1:400 dilution), anti-human Granzyme B-Pacific Blue (Biolegend, 1:400 dilution) and 1 μl of human Fc receptor blocking solution (Human TruStain FcX, Biolegend) for 30 minutes at room temperature, washed and resuspended in staining buffer before acquisition on LSR Fortessa 4 (Becton Dickinson) with Diva software. FlowJo software version 6.0 was used for gating.
3
Multiparameter Flow Cytometry of Hematopoietic Cells
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50 μL peripheral blood or ~5×105 bone marrow cells in PBS with 10% FBS were blocked with anti-mouse CD16/CD32 (cat # 101302, Biolegend, San Diego, CA, USA) and then stained with fluorophore conjugated antibodies. Following staining, red blood cells were lysed with RBC lysis buffer (cat # sc-296258, Santa Cruz Biotechnology, Dallas, TX, USA) for 10 min and washed twice with 10% FBS in PBS. Peripheral blood samples underwent a second 5 min RBC lysis prior to washing. Samples were acquired on a BD Accuri C6 or Beckman coulter Gallios flow cytometer and analyzed using BD Accuri Csampler software or FlowJo. Cells were stained with anti-human CD34 PE (cat # 343606, Biolegend), anti-human CD11b PE (cat # 555388, BD Biosciences, San Jose, CA, USA), anti-human CD3 PE/Dazzle™ 594 (cat # 300450, Biolegend), anti-mouse CD45 PerCP-Cy5.5 (cat # 103132 Biolegend), anti-mouse CD45 PerCP (cat # 557235, BD), anti-human CD19 PE-Cy7 (cat # 302216, Biolegend), anti-human CD45 APC (cat # 555485, BD Bioscience or cat # 304012, Biolegend). Analysis based 10 000 bone marrow and 3 000 blood cells from the live cell gate.
4
Immunoglobulin and Nanoparticle Characterization
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Privigen® human immunoglobulin from CSL Behring (King of Prussia, PA, USA) was used for flow cytometry. Anti-human-CD14-APC-Cy7 and anti-human-CD19-PE-Cy7 were from BioLegend (San Diego, CA, USA), anti-human-CD3-Pacific-blue and Annexin V/7AAD apoptosis kits were from BD Biosciences (San Jose, CA, USA), and anti-human-CD15-APC was from Miltenyi Biotech GmbH (Bergisch Gladbach, Germany). Lectin from Phaseolus vulgaris (PHA-L) and lipopolysaccharide (LPS) from Sigma-Aldrich Co. (St Louis, MO, USA) were used for immune cell stimulation. The commercially available MRI contrast agent ferumoxsil (Lumirem®; Guerbet, Villepinte, France) was used as a negative control for the final comparative analysis of different NP preparations.
5
Multiparameter Flow Cytometry of Hematopoietic Cells
6
Multiparametric Flow Cytometry for Immune Cell Phenotyping
7
Isolation and Characterization of SARS-CoV-2 Spike-Reactive B Cells
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Human PBMCs were isolated via Ficoll density gradient centrifugation, and B cells were enriched using a B cell enrichment kit from Stemcell following the manufacturer’s protocol. Cells were then incubated with 200 μL APC-conjugated S protein trimer (Invitrogen, catalog A20186) and Alexa Fluor 488–conjugated S1 subunit (Invitrogen, catalog A20181) in combination with PE-Cy7 anti–human CD19 (BioLegend, catalog 302216) and APC-Cy7 anti–human CD20 (BioLegend, catalog 302314). Cells were stained in staining buffer (PBS plus 2% FBS) for 30 to 60 minutes on ice and then washed with 15 mL ice-cold staining buffer. Next, stained samples were sorted into cell lysis buffer in 96-well plates via a FACSAria II Cell Sorter (BD Biosciences). CD19+CD20+S trimer+S1+ live cells within the lymphocyte gate determined by forward scatter (FSC) and side scatter (SSC) were collected. The 96-well plates were then immediately frozen at –80°C for future use.
8
Isolation of Human CD14+ Monocytes
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Human CD14+ monocytes were isolated from human buffy coats collected from healthy donors at the Blutspendezentrum Basel as follows. Approximately 50–60 mL of buffy coat were diluted with 70 mL of PBS. Ficoll-Paque (VWR, cat# 17–1440) was added to the botton of Sepmate-50 tubes (StemCell Technologies; cat# 85450) for density gradient centrifugation. Diluted blood was slowly added on top of the Ficoll-Plaque and tubes were spun at 1400 rpm for 25 min with slow acceleration and no brakes. The cell mononuclear fraction was collected and placed in separated tubes. Two washing steps were performed with PBS 2% FBS 0.5 M EDTA before initiating the purification (positive selection) of human CD14+ cells using CD14 MicroBeads (Milteny Biotec; cat# 130-050-201) and LS columns (Miltenyi Biotec; cat# 130-042-401) according to manufacturer's instructions. Cell purity was checked by FACS staining for BV785 anti-human CD14 (BioLegend; cat# 301840), Alexa Fluor 488 mouse anti-human CD3 (BD Pharmigen; cat# 557694), PE/Cy7 anti-human CD19 (BioLegend; cat# 302215) and PE anti-human CD66b (BioLegend; cat# 305106).
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