Blood samples were obtained after the approval from the Beijing Institute of Basic Medical Sciences, consent from 9 normal human subjects, 3 patients with MM, and 6 patients with SLE from Clinical Trial Center (Beijing 301 Hospital). CD19+ B cells were isolated using human CD19 MicroBeads (Cat No. 130-090-880, Miltenyi Biotec).
Human cd19 microbeads
Manufactured by Miltenyi Biotec
Sourced in Germany, United States
The Human CD19 MicroBeads are magnetic beads coated with antibodies specific for the CD19 antigen expressed on human B cells. They are used for the isolation and enrichment of CD19-positive cells from a heterogeneous cell population.
Automatically generated - may contain errors
Lab products found in correlation
Human b cell isolation kit 2, by Miltenyi Biotec (2 mentions)
Ficoll paque, by GE Healthcare (2 mentions)
Facsaria fusion, by BD (2 mentions)
Human cd34 hematopoietic stem cells hscs, by STEMCELL (2 mentions)
Ficoll hypaque, by GE Healthcare (2 mentions)
Pan t cell isolation kit, by Miltenyi Biotec (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Aria 2, by BD (1 mentions)
Lymphoprep, by Abbott (1 mentions)
Sepmate 50, by STEMCELL (1 mentions)
Percp cy5, by BioLegend (1 mentions)
Bv605, by BioLegend (1 mentions)
Human il 4, by Thermo Fisher Scientific (1 mentions)
Human cd138 microbead, by Miltenyi Biotec (1 mentions)
Erythrocyte lysis buffer, by Qiagen (1 mentions)
Ficoll histopaque, by Merck Group (1 mentions)
Facscan, by BD (1 mentions)
Ficoll paque plus, by GE Healthcare (1 mentions)
Facsaria, by BD (1 mentions)
Anti cd27 apc cy7, by BioLegend (1 mentions)
18 protocols using human cd19 microbeads
1
Isolation of CD19+ B cells
2
Isolation and Purification of CLL Cells
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Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood by density gradient centrifugation using Ficoll-Paque (GE Healthcare, Chicago, IL, USA). CD19+ B cells, including MEC-1 cells, were purified by magnetic-activated cell sorting (MACS) using human B-cell isolation kit II or human CD19 MicroBeads (Miltenyi, Bergisch-Gladbach, Germany). Purification of primary CLL cells (CD19+ CD5+) and MEC-1 cells (GFP, CD19+) was performed using fluorescence-activated cell sorting (FACS) (BD Aria II, BD Bioscience, Franklin Lakes, NJ, USA). All antibodies are listed in Supplementary Table 3 . Peripheral blood, spleen, and axillary lymph nodes were harvested per mouse. Organs were meshed through a 70 μm cell strainer in PBS buffer and erythrocytes were lysed using G-DEXTMIIb RBC Lysis Buffer (Intron Biotechnologies).
3
Primary B Cell Expansion Protocol
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125 ml blood samples were processed within several hours of being drawn with EDTA to prevent clotting by TSRI normal blood donor services. PBMCs were purified by density gradient centrifugation on Lymphoprep (Alere technologies) in SepMate-50 (Stem cell technologies) tubes. B cells were positively purified from ACK treated, PBS washed PBMCs using human CD19 Microbeads (Miltenyi) according to the manufacturer’s instructions. Purified CD19 +B cells were immediately placed into culture at 0.5 × 106 cells/ml. Primary B cells were cultured in CD40L/IL-4 B cell expansion media (Miltenyi), with antibiotics (pen/strep), according to the manufacturer’s instructions. Cells were kept between 0.5 and 1.5 × 106 cells/ml at 37°C in a humidified incubator with 5.0% CO2. Media was replaced every four days and B cell clusters disrupted by pipetting at the time of media replacement. CFSE staining (CellTrace) was performed according to the manufacturer’s instructions.
4
Enrichment and Sorting of SARS-CoV-2 Specific Memory B Cells
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B cells, enriched from PBMCs with human CD19 MicroBeads (Miltenyi), were incubated with 2 μg/ ml flag-tagged S protein or mixture of flag-tagged S protein (Genscript, Cat. Z03481) and His-tagged RBD (Chen et al., 2020 (link)) on ice for 30 min. Cells were then washed with 2% fetal bovine serum (FBS) (Hyclone) in PBS and stained with mixture of anti-human IgG (Percpcy5.5; Biolegend Cat. 410710), anti-human IgD (FITC; Biolegend Cat. 348205), anti-human IgM (Bv605; Biolegend Cat. 314524), anti-CD27 (APCcy7; Biolegend Cat. 356404). A mixture of PE- and APC-conjugated anti-flag antibodies (Biolegend Cat. 637309 and 637307) was also added for gating S-specific double positive cells, or a mixture of PE-conjugated anti-His (Biolegend Cat. 362603) and APC-conjugated anti-flag, for S positive and RBD negative cells. Memory B cells were gated on DAPI-CD19+IgM-IgD-IgG+CD27+. Individual S double positive or S+RBD- cells were sorted with a FACSAria Fusion (BD Biosciences) into each well of 96-well microplates containing 4 μl/well of lysis buffer (0.5X PBS, 10 mM dithiothreitol, and 4U RNaseOUT). Lysed cells were immediately frozen and stored at −80°C until use.
5
Autologous B Cell Culture and Expansion
6
Isolation of BM CD19+ and CD138 Cells
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We collected samples from all 56 subjects and we isolated mononuclear cells (MNCs) from the BM blood samples (range, 4–6 mL) using Ficoll density gradient centrifugation at 800 rpm for 20 min. Immediately after, we selected BM CD19+ cells using Human CD19 MicroBeads; afterward, CD138 cells were positively isolated from the collected CD19− cells using Human CD138 Micro Beads according to the manufacturer’s instructions (Miltenyi Biotec, Milan, Italy) [17 (link)].
7
Isolation and Characterization of B-cell Subsets
8
Fractionation and Purification of Blood Cell Types
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Citrated peripheral blood was collected and fractionated over the Ficoll-Histopaque (Sigma, St. Louis, MO, USA). The platelet rich plasma (PRP) layer was removed and platelets were pelleted and resuspended in Beads Buffer (BB; PBS with 0.5% w/v of bovine serum albumin and 2.5 m Methylenediaminetetraacetic acid final concentration). Leukocytes were removed with MACS Human CD45 microbeads reagents (Miltenyi Biotec, Auburn, CA, USA) [39] . The mononuclear cell layer was recovered, washed and re-suspended in BB for isolation of T-cells and B-cells using human CD3 and human CD19 microbeads (MiltenyiBiotec, Auburn, CA, USA), respectively. The buffy coat atop the red blood cells was removed, washed with PBS, treated with Erythrocyte lysis buffer (Qiagen, Hilden, Germany), pelleted and resuspended in BB. Granulocytes were isolated using MACS Human CD15 microbeads (MiltenyiBiotec). Lastly, erythrocytes were isolated from the lowest Ficoll-Histopaque layer by double immunodepletion of white blood cells and granulocytes using Human CD45 and CD15 microbeads. Cell purity was assessed on a FACScan (Becton Dickinson, Franklin Lakes, NJ, USA) using FlowJo 8.5.3 software (Tree Star Inc., Ashland, OR, USA).
9
Isolation and Characterization of Cell Samples for CLL Research
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Healthy blood from anonymous donors were obtained with written consent from the Department of Transfusion Medicine, NIH Clinical Center, and analyzed with the approval of the ethical review committee of the NIH. CLL patient samples were obtained after written informed consent in accordance with the Declaration of Helsinki, applicable federal regulations, and requirements from the National Heart, Lung, and Blood Institute Institutional Review Board. The clinical study is registered at clinicaltrials.gov as NCT00923507 and NCT00071045 . Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats using Ficoll-Hypaque (GE Healthcare Biosciences, Pittsburgh, PA) by gradient centrifugation. The following cell lines were cultured in complete media and used as a target in assays: CCRF-CEM, MEC1, Nalm6 and U937. MEC1-001 and MEC1-002 were descripted previously (17 (link)), and were tested negative for mycoplasma contamination. Both B- and T- cells were isolated from buffy coat using human CD19 microbeads and pan T cell isolation kit (Miltenyi Biotech, Auburn, CA). Human CD34+ hematopoietic stem cells (HSCs) were purchased from StemCell Technologies (Vancouver, BC, Canada). MEC1-001-Siglec-6 Transgenic (TG) cells we generated by retroviral transduction with pEV-Thy1.1-hSiglec-6. The CD19-CAR construct corresponds to the previously described MSGV-FMC63-28Z (19 (link)).
10
Isolation and Sorting of T Cell Subsets
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All experiments using primary cells were conducted at the MSKCC, using institutional-review-board (IRB)-approved protocols. PBMCs were isolated from whole blood of two different healthy donors using Ficoll-Paque PLUS (GE Healthcare, Chicago, IL, USA). B cells were separated from PBMCs by using human CD19 microbeads, according to the manufacturer’s manual (Miltenyi Biotec, Bergisch Gladbach, Germany). The B cell-depleted PBMCs were then subjected to fluorescence-activated cell sorting (FACS) using a FACSAria (BD Biosciences, San Jose, CA, USA) high-speed cell sorter to obtain CD5+ cells. These cells were then sorted to collect CD4+ and CD8+ T cell populations that were subsequently pooled together to be used in experiments.
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