Peripheral Blood Mononuclear cells (PBMCs) were isolated from heparinized peripheral blood using Ficoll–Paque gradient centrifugation (Sigma-Aldrich, St. Louis, MO, USA).
Ficoll paque gradient centrifugation
Manufactured by Merck Group
Sourced in United States
Ficoll–Paque gradient centrifugation is a laboratory technique used for the separation and purification of cells, organelles, and other biological materials based on their density. It employs a density gradient medium to facilitate the separation process during centrifugation.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Cd4 dynabeads, by Thermo Fisher Scientific (1 mentions)
Detachabead cd19, by Thermo Fisher Scientific (1 mentions)
Dynabeads cd25, by Thermo Fisher Scientific (1 mentions)
Dynabead, by Thermo Fisher Scientific (1 mentions)
Sodium heparin vacutainer, by BD (1 mentions)
Dextran sedimentation, by Merck Group (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Rpmi8226, by American Type Culture Collection (1 mentions)
4 protocols using ficoll paque gradient centrifugation
1
Isolation of PBMCs from Blood
2
Isolation of B Cells and T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized peripheral blood using Ficoll–Paque gradient centrifugation (Sigma-Aldrich, St. Louis, MO, USA). CD19+ B cells were isolated by magnetic bead-based positive selection using Dynabeads and DETACHaBEAD CD19 (Invitrogen). CD4+ T cells were isolated with Dynabeads CD4 (invitrogen) and CD25+ T cells were depleted from the CD4+ T cell population using Dynabeads CD25 (invitrogen). All isolations were done according to the manufacturer’s instructions. The purity of isolated CD19+ B cells was consistently >95% as analyzed by flow cytometry.
3
Neutrophil Isolation and Stimulation Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Venous blood from healthy adult volunteers (Children’s and Women’s Research Ethics Board of the University of British Columbia certificate number H15-00351) was collected in sodium heparin vacutainers (BD Bioscience, San Jose, CA, USA). Polymorphonuclear cells (PMN) were isolated using dextran sedimentation (Sigma-Aldrich, Saint Louis, MO, USA) and Ficoll-Paque gradient centrifugation as described (26 (link)). Neutrophils consistently comprised no less than 95% of the PMN population (pocHi 100i™ Hematology Analyzer, Sysmex Corp., Kobe, Japan), thus PMN are referred to as neutrophils throughout this study. Isolated neutrophils were suspended in complete RPMI (RPMI 1640 with 10% fetal bovine serum (FBS), 2 mM L -glutamine and 1 mM sodium pyruvate; all from ThermoFisher Scientific, Waltham, MA, USA) and used immediately. Alternatively, neutrophils were kept on ice in Krebs-Ringer Glucose buffer (KRG, pH 7.3) containing 1 mM Ca2+ for a maximum of 6 h prior to use in downstream experiments. Unless otherwise stated, neutrophils (5 x 106/mL) were incubated at 37°C, 5% CO2 in the absence and presence of HA (200 μg/mL) and/or TNFα (10 ng/mL) for 20 min.
4
Characterization of Multiple Myeloma Cell Lines and Primary Bone Marrow Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell lines and primary BM cells MM cell lines: CAG and ARK (established at the Arkansas Cancer Research Center), RPMI-8226 (CCL 155, ATCC), U266 (TIB-196, ATCC) and ARP-1. All cell lines were negative for mycoplasma contamination. Bone marrow (BM) aspirates were obtained upon written consent from a total of 40 patients at various disease stages (clinical data are presented in Supplementary Table 1, see section on supplementary data given at the end of this article). Additionally, one normal BM sample from healthy subject was collected. All BM aspirates were obtained from patients treated at the Meir Medical Center after approval by an Institutional Review Board (IRB number #180-2010) . BM mononuclear cells were isolated by Ficoll-Paque gradient centrifugation according to the manufacturer's instructions (Sigma-Aldrich). The different mononuclear cell populations were characterized and immunophenotyping was performed by flow-cytometry. Cells were grown in RPMI-1640 medium supplemented with 10% heat-inactivated FCS, penicillin and streptomycin antibiotics and the passage number for the experiments described was up to 20.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!