Peripheral blood mononuclear cells (PBMCs) were isolated from human blood (i.e., buffy-coats) using Lymphoprep-TM (StemCell Technologies, Vancouver, BC, Canada) gradient centrifugation. From the selected (PBMC)-pool, CD14+ monocytes were isolated using magnetic CD14+ Microbeads (Cat. no. 130-050-201; Miltenyi Biotec, Bergisch Gladbach, Germany). Purity and recovery of CD14+ monocytes were determined by CD14-FITC antibody labeling (Cat. no. 130-113-708; Miltenyi Biotec), whereas cell viability was determined by propidium iodide (PI) staining and subsequent flow cytometry analysis on a BD FACSAria III (BD Biosciences, San Jose, CA, USA). For the generation of macrophages, CD14+ monocytes were cultured in macrophage growth medium (RPMI 1640 with 10% FBS, 1% streptomycin/penicillin and 100 ng/mL of macrophage colony-stimulating factor (M-CSF); (Cat. no. 300-25; PrepoTech, Rocky Hill, NJ, USA) and kept in a humidified atmosphere (5% CO2, 37 °C), for 6 days. The incubation medium was replaced every three days with fresh (pre-warmed) medium.
Lymphopreptm
Manufactured by STEMCELL
Sourced in Canada, United Kingdom, France, United States, Germany, Norway
LymphoprepTM is a sterile density gradient media used for the separation and isolation of mononuclear cells from whole blood, bone marrow, or other cell suspensions by centrifugation.
Automatically generated - may contain errors
Lab products found in correlation
L glutamine, by Merck Group (5 mentions)
Sepmate tube, by STEMCELL (4 mentions)
Rpmi 1640, by Merck Group (4 mentions)
Penicillin, by Thermo Fisher Scientific (4 mentions)
Human ab serum, by Merck Group (3 mentions)
Labtekstm, by Thermo Fisher Scientific (3 mentions)
Non treated cell culture plates, by Corning (2 mentions)
Phytohemagglutinin l, by Thermo Fisher Scientific (2 mentions)
Pen strep, by Merck Group (2 mentions)
Fetal calf serum (fcs), by Merck Group (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Sepmatetm tube, by STEMCELL (2 mentions)
Rnalater, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Facsaria 3, by BD (1 mentions)
Lymphoprep, by Miltenyi Biotec (1 mentions)
Ls column, by Miltenyi Biotec (1 mentions)
Rosettesep ctc enrichment cocktail containing anti cd56, by STEMCELL (1 mentions)
70 protocols using lymphopreptm
1
Isolation and Differentiation of Human Monocyte-Derived Macrophages
2
Isolation of Peripheral Blood CD4+ T Cells
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lymphoprepTM (STEMCELL Technologies) was used to isolate peripheral blood mononuclear cells (PBMCs) from buffy coat or heparinized blood obtained from the blood bank (UNN, Tromsø Norway), according to the manufacturer’s instructions. Enriched CD4+ T cells were obtained by negative selection from lymphoprep-PBMCs pellets using the magnetic CD4+ T cell human isolation kit (130-096-533 Miltenyi Biotec) and LS Column (Miltenyi Biotec).
3
Peripheral Blood Mononuclear Cells and Circulating Tumor Cells Isolation
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PBMCs isolation was performed from 10 mL of peripheral blood (collected using an EDTA tube) by density gradient centrifugation protocol (LymphoprepTM, STEMCELL Technologies, Vancouver, BC, Canada) in SepMate™ tubes (STEMCELL Technologies, Vancouver, BC, Canada) according to manufacturer’s instructions. Another 10 mL of peripheral blood was used to collect CTCs by negative selection using the RosetteSep™ CTC Enrichment Cocktail Containing Anti-CD56 (STEMCELL Technologies, Vancouver, BC, Canada) according to the manufacturer’s protocol. Enriched cells were placed in RNAlaterTM Solution (Invitrogen, ThermoFisher Scientific, Carlsbad, CA, USA) and kept at −80 °C until further analyses.
4
Isolation and Purification of Human PBMCs
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Human peripheral blood mononuclear cells (PBMCs) were isolated in LymphoprepTM and SepMateTM RUO tubes (STEMCELL Technologies, United States) by using density gradient centrifugation. Then, 2.5 ml of blood was diluted with an equal amount of PBS with 2% fetal bovine serum. The blood was layered on top of 5 ml LymphoprepTM, being careful to minimize the mixing of blood with LymphoprepTM. The tubes were centrifuged at 800 × g for 20 min at room temperature with brake-off. The upper plasma layer was removed and discarded without disturbing the plasma–LymphoprepTM interface. The mononuclear cells (MNC) layer was removed and retained at the interface. The MNCs were washed once with RPMI1640 medium. The monocytes were separated from other leukocytes by adherence to plastic after being cultured in the plate for 2 h. The monocytes were collected for mRNA detection by RT-PCR (n = 3/group).
5
Isolation and Expansion of Human CD8+ T Cells
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Human peripheral blood mononuclear cells (PBMCs) were isolated from healthy donor blood using LymphoprepTM (catalogue no. 07801, Stem-Cell Technologies Cambridge UK) and following the recommended original company’s protocol. CD8+ lymphocytes were isolated from PBMCs using CD8 Micro Beads (catalogue no. 130-045-201, Miltenyi Biotec, Bergisch Gladbach, Germany). CD8+ cells were propagated in RPMI 1640 medium containing 10% FBS, 1% penicillin/streptomycin, human IL-2 (210 U/mL), anti-CD3 (1 μg/mL) and anti-CD28 (1 μg/mL) antibodies in 37 °C with 5% CO2 incubator for 2–7 days.
6
Extracting Extracellular Vesicles from Plasma
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Plasma samples (2 mL) were thawed on ice, and EVs were isolated using an exoRNeasy Plasma Midi Kit (Qiagen, #77044). PBMCs were extracted from the whole blood of DLBCL patients using density gradient medium (LymphoprepTM, STEMCELL Technologies, #07851).
7
Isolation and Differentiation of Human Monocyte-Derived Macrophages
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Peripheral blood buffy coats were obtained from the Irish Blood Transfusion Services in Dublin, Ireland. PBMCs were isolated by density gradient centrifugation with LymphoprepTM (Stemcell Technologies, Vancouver, BC, Canada) and seeded at 2.5 × 106 cells/mL in Roswell Park Memorial Institute (RPMI) 1640 medium (Bio-Sciences, Nottingham, UK), supplemented with 10% AB-human serum (Sigma-Aldrich, St. Louis, MO, USA) and plated onto non-treated cell culture plates (Corning). LabTeksTM (Nunc, Roskilde, Denmark) were also seeded to determine the multiplicity of infection (MOI; see Section 4.2 ). To obtain hMDMs, the cells were cultured over 7 days at 37 °C and 5% CO2 to allow differentiation prior to experimentation. Cells were washed every 2–3 days to remove non-adherent cells. On day 7, hMDMs were routinely greater that 90% pure, as determined by flow cytometry-based analysis of CD14 and CD68 co-expression (data not shown).
8
Isolation of Peripheral Blood Cells
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Donor blood (collected as part of ethical approval 033–10) was diluted with an equal volume of 2% Dextran (D8802, Sigma) in phosphate buffered saline pH7.4 (PBS; 10010, Gibco) and incubated until red blood cells (RBCs) had sedimented. Granulocytes and peripheral blood mononuclear cells (PBMCs) were isolated from the phase above the red blood cells by density gradient centrifugation using LymphoprepTM (07851, STEMCELL Technologies) as per the manufacturer’s instructions. In brief, the blood was layered onto an equal volume of LymphoprepTM and then centrifuged at 850g for 20 minutes at room temperature. The PBMC fraction was transferred to a new tube and washed once with buffer (PBS containing 2 mM EDTA and 2% heat inactivated FBS (15575, 10438, Gibco)) before isolation of monocytes. The granulocyte pellet was kept on ice and resuspended into 0.5 mL of cold PBS and 6.0 mL of water added to lyse the remaining RBCs. The lysis reaction was terminated after 15 seconds with stopping buffer (hypertonic PBS, 410 mM NaCl, pH7.4).
9
CMML Patient Sample Collection and Cell Line Processing
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Chronic myelomonocytic leukemia patient samples were collected at the Division of Hematology, Medical University of Graz, Austria, as well as in the Austrian Biodatabase for CMML. All samples were processed and stored as described in detail in the Online Supplementary Methods. Healthy CD34+ HSPC were collected from umbilical cord blood specimens (EasySep, STEMCELL Technologies) according to the manufacturerś instructions and processed as described before.24 (link) Peripheral blood samples from healthy donors were used to collect CD14+ monocytes (MACS, Miltenyi Biotec), B lymphocytes and granulocytes (LymphoprepTM, STEMCELL Technologies and human B Lymphocyte enrichment set, BD biosciences) according to the manufacturer’s protocol. 293T, NB4 and HL-60 cell lines were obtained from the German National Resource Center for Biological Material (DSMZ, Braunschweig, Germany). Low passage stocks were frozen and cells were always passaged for less than six months after resuscitation. Additionally, cells were screened by variable number of tandem repeat profiling (VNTR) for authenticity.21 (link) Lentiviral transduction of cell lines and primary HSPC were performed as previously described.12 (link),21 (link),22 (link)
10
Stimulating Donor PBMCs with Tumor Conditions
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Healthy donor PBMCs were prepared from whole blood collected in EDTA Vacutainer tubes (BD Biosciences) by density gradient centrifugation over LymphoprepTM (Stemcell Technologies). PBMCs were plated at a concentration of 1 × 106 cells per ml in complete RPMI media (10% FBS, 1% penstrep) and activated using plate bound anti-CD3 (10 μg/ml, Biolegend, USA) and anti-CD28 (10 μg/ml, Ancell, USA). PBMCs were concomitantly activated in the absence or presence of 50% OGJ cell conditioned media from 48 h vehicle-treated OE33/SK-GT-4 cells or FLOT, CROSS CT or MAGIC chemotherapy-treated OE33/SK-GT-4 cells or 50% tumour conditioned media (conditioned media) for 48 h. PBMCs were then harvested and stained for flow cytometry.
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