B lymphocytes expressing CD19+ were negatively selected from peripheral blood of patients immediately after blood withdrawal using RosetteSep B-lymphocyte enrichment kit (STEMCELL Technologies, Vancouver, BC, Canada). The purity of the cell preparations was measured using flow cytometry (FC500; Beckman Coulter, Pasadena, CA, USA) based upon cell surface co-expression of CD19 and CD5. Purity was always greater than 99%. CLL cells were CD19+CD5+, T cells were CD3+, and B cells were CD19+CD5−. Preparations were virtually devoid of natural killer (NK) cells, T lymphocytes, and monocytes. Total peripheral blood mononuclear cells (PBMCs) and purified leukemic cells were cultured in parallel in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, and 15 μg/ml gentamicin (Sigma-Aldrich, Carlsbad, CA, USA). Lymphocytes were cultured at a concentration of 1 × 106 cells/500 μl in the presence or absence of different stimuli or inhibitors, as indicated. HDL NP were added to the samples at a concentration of 30 nM and 100 nM and allowed to incubate for up to 72 hours in vitro.
Rosettesep b lymphocyte enrichment kit
Manufactured by STEMCELL
Sourced in Canada
The RosetteSep B-lymphocyte enrichment kit is a laboratory product designed to isolate and enrich B-lymphocytes from whole blood or buffy coat samples. The kit uses a proprietary combination of antibodies and magnetic particles to selectively label and remove unwanted cells, leaving the desired B-lymphocytes for further analysis or experimentation.
Automatically generated - may contain errors
Lab products found in correlation
Fc500, by Beckman Coulter (3 mentions)
Rpmi 1640 medium, by Euroclone (2 mentions)
Gentamicin, by Merck Group (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Easysep cell isolation kit, by STEMCELL (1 mentions)
Dmem medium, by Thermo Fisher Scientific (1 mentions)
Lymphoprep, by STEMCELL (1 mentions)
Hs5 human bone marrow stromal cell line, by American Type Culture Collection (1 mentions)
Blasticidin, by Thermo Fisher Scientific (1 mentions)
Mec 1 cell line, by Leibniz Institute DSMZ (1 mentions)
Navios, by Beckman Coulter (1 mentions)
5 protocols using rosettesep b lymphocyte enrichment kit
1
Enrichment and Stimulation of CLL Cells
2
Isolation and Characterization of Leukemic CD19+ Cells
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Leukemic CD19 cells were negatively selected after blood withdrawal using the Rosette Sep B-lymphocyte enrichment kit (Stem Cell Technologies). Purity of the preparation was more than 99% and cells co-expressed CD19 and CD5 on their cell surfaces, as shown by flow cytometry (FC500; Beckman Coulter); the preparation was virtually devoid of natural killer (NK) cells, T lymphocytes, and monocytes [14 (link)].
Cells were cultured in RPMI 1640 medium (Invitrogen) supplemented with 10% volume/volume (v/v) Fetal Bovine Serum (FBS) and 15 mg/ml Gentamicin (complete RPMI) at 37°C, 5% CO2. The morphology of neoplastic population was evaluated on cytocentrifuged cells stained with Haematoxylin and Eosin.
Cells were cultured in RPMI 1640 medium (Invitrogen) supplemented with 10% volume/volume (v/v) Fetal Bovine Serum (FBS) and 15 mg/ml Gentamicin (complete RPMI) at 37°C, 5% CO2. The morphology of neoplastic population was evaluated on cytocentrifuged cells stained with Haematoxylin and Eosin.
3
Isolation of Leukemic Lymphocytes from CLL Patients
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Leukemic lymphocytes were obtained from peripheral blood of CLL patients, diagnosed according to the updated National Cancer Institute Working Group (NCIWG) guidelines99 (link). All patients were either untreated or off therapy for at least 6 months before the beginning of the study. Leukemic CD19 cells were negatively selected from fresh peripheral blood using RosetteSep B-lymphocyte enrichment kit (StemCell Technologies). Purity of all preparations was always more than 99%, and the cells co-expressed CD19 and CD5 on their cell surfaces as checked by flow cytometry (FC500; Beckman Coulter); preparations were virtually devoid of natural killer (NK) cells, T lymphocytes, and monocytes. Patients have been divided in GOOD prognosis and POOR prognosis based on clinical and biological parameters.
4
Isolation and culture of primary B cells
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The HS5 human bone marrow stromal cell line was obtained from the American Type Culture Collection (ATTC) and cultured in DMEM medium (GIBCO) supplemented with 10% v/v FBS, 15 mg/mL Pen/Strep at 37°C and 5% CO2. Primary leukaemic, from patients, and healthy, from donors, CD19+ B cells were negatively selected from fresh PB and BM using the RosetteSep B lymphocyte enrichment kit (Stemcell Technologies) and separated via density gradient Lymphoprep (Stemcell Technologies). Healthy B cells were then isolated with EasySep Cell isolation kit (Stemcell Technologies). B cells were isolated from lymphoid tissues after mechanical smashing to recover the cells in suspension and later purified following the same protocol described for healthy B cells. The purity of all the isolated B cells was >98%. Primary cells were cultured in RPMI 1640 medium (EuroClone) supplemented with 10% (v/v) Fetal Bovine Serum (FBS) and 15 mg/mL Pen/Strep (complete RPMI) at 37°C and 5% CO2.
5
Isolation and Cultivation of CD19+ Cells
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CD19 cells were negatively selected from fresh pheripheral blood from patients or healthy donors using the RosetteSep B-lymphocyte enrichment kit (StemCell Technologies, Vancouver, Canada). Human healthy cells were further negatively selected using B-lymphocyte enrichment kit (StemCell Technologies, Canada). The purity of all preparations was always higher than 99%, and the cells co-expressing CD19 and CD5 on their surface as assayed by flow cytometry (Navios Beckman Coulter Life Science, Indianapolis IL, United States) preparations were virtually devoid of natural killer cells, T lymphocytes, and monocytes.
The MEC1 cell line was obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) and cultured in RPMI 1640 medium (Euroclone, Milan, Italy) supplemented with 10% volume/volume (v/v) fetal bovine serum (FBS) and 15 mg/ml gentamicin (complete RPMI) at 37°C and 5% CO2. MEC1-HS1-YFP was generated by transfecting MEC1 cells, taking advantage of Nucleofector Technology (AMAXA) with the use of the program X-001, solution V.
The Vivid ColorsTM pcDNATM6.2/N-YFP-DEST Vector expressing the HS1 gene (Thermo Fisher Scientific, Waltham, MA, United States) was used for transfection and blasticidin (Thermo Fisher Scientific, United States) for the antibiotic selection.
The MEC1 cell line was obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) and cultured in RPMI 1640 medium (Euroclone, Milan, Italy) supplemented with 10% volume/volume (v/v) fetal bovine serum (FBS) and 15 mg/ml gentamicin (complete RPMI) at 37°C and 5% CO2. MEC1-HS1-YFP was generated by transfecting MEC1 cells, taking advantage of Nucleofector Technology (AMAXA) with the use of the program X-001, solution V.
The Vivid ColorsTM pcDNATM6.2/N-YFP-DEST Vector expressing the HS1 gene (Thermo Fisher Scientific, Waltham, MA, United States) was used for transfection and blasticidin (Thermo Fisher Scientific, United States) for the antibiotic selection.
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