Primary human peripheral blood mononuclear cells (PBMCs) from anonymous, healthy donors (New York Blood Center) were isolated by Ficoll gradient separation as previously described (Reyes-Robles et al., 2016 (link)) CD14+ monocytes were then isolated from the PBMC fraction by positive selection using the EasySep™ Human CD14 Positive Selection Kit II (STEMCELL Technologies) according to the manufacturer’s protocol. Cells were plated at 1×106/mL in RPMI supplemented with 10% FBS, 10 mM HEPES, 100 U/mL penicillin, 100 μg/mL streptomycin and 50 ng/mL GM-CSF. The cells were incubated at 37°C with 5% CO2. Media was replenished with GM-CSF on day 2. Cells were harvested and plated for infection on day 4.
Easysep human cd14 positive selection kit 2
Manufactured by STEMCELL
Sourced in Canada, United Kingdom
The EasySep Human CD14 Positive Selection Kit II is a laboratory product designed to isolate CD14-positive cells from human samples. It utilizes a fast and easy-to-use magnetic separation method to quickly enrich for the desired cell population.
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Lab products found in correlation
Ficoll paque plus, by GE Healthcare (4 mentions)
Recombinant human il 4, by Thermo Fisher Scientific (3 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (2 mentions)
Easysep human na ve cd4 t cell isolation kit 2, by STEMCELL (1 mentions)
Ficoll hypaque gradient centrifugation, by Cytiva (1 mentions)
Human rankl, by Thermo Fisher Scientific (1 mentions)
Recombinant human m csf, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Trypan blue, by Thermo Fisher Scientific (1 mentions)
Lymphoprep centrifugation, by STEMCELL (1 mentions)
Interleukin 4 (il 4), by Immunotools (1 mentions)
Rpmi 1640, by Cytiva (1 mentions)
Gm csf, by Immunotools (1 mentions)
Recombinant human granulocyte macrophage colony stimulating factor gm csf, by Thermo Fisher Scientific (1 mentions)
Mouse anti human cd47 clone b6h12, by Thermo Fisher Scientific (1 mentions)
Mouse igg1, by Thermo Fisher Scientific (1 mentions)
Non enzymatic cell dissociation buffer, by Merck Group (1 mentions)
Ficoll paque premium, by GE Healthcare (1 mentions)
Lsrfortessa x 20, by BD (1 mentions)
26 protocols using easysep human cd14 positive selection kit 2
1
Isolation and culture of human monocytes
2
Isolation of CD14+ Monocytes and Naive CD4+ T Cells
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Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats (Etablissement Français du Sang) by Ficoll-Hypaque gradient centrifugation (specific gravity, 1.077 g/mL; Amersham Biosciences, USA) density.CD14+ cells were positively separated by anti-CD14 magnetic beads according to the Manufacturer’s instructions (EasySep™ Human CD14 Positive Selection Kit II, Stemcell technologies). Isolated cells routinely contained more than 95% of CD14+ cells. Naive CD4+ T cells were isolated from PBMCs using EasySep™ Human Naïve CD4+ T Cell Isolation Kit II (Stemcell technologies). The cell population obtained, contained >95% of CD3+ CD4+ CD45RA+ CD45RO− cells, as assessed by flow cytometry.
3
Isolation of CD14+ Monocytes from Blood
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Peripheral blood mononuclear cells (PBMCs) were obtained from healthy donor blood by density gradient centrifugation in a Ficoll-Paque PLUS (GE Healthcare, Chicago, IL, USA). CD14+ cells were then isolated using the EasySep™ Human CD14-Positive Selection Kit II (STEMCELL Technologies, Vancouver, BC, Canada) according to the manufacturer’s protocols.
4
Isolation and Osteoclastogenesis of CD14+ Cells
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Buffy coats were received from the Scottish National Blood Transfusion Service. Magnetic selection was performed using EasySep Human CD14 positive selection kit II (product no. 17858, Stemcell Technologies, Cambridge, UK. The cells were re-suspended at 1 × 106 cells/ml in α-MEM (alpha minimum essential media) supplemented with 10% FBS, 0.02 mM l -glutamine, 10 U/ml Penicillin, and 0.1 μg/ml Streptomycin (all components from Sigma-Aldrich, Dorset, UK). Following suspension at 1 × 106 cells/ml, 25 ng/ml of recombinant human M-CSF (product no. 300-25, Peprotech, London, UK) was added. Cells were plated in 24 well plates and incubated overnight at 37 °C and 5% CO2. After approximately 18 h incubation 25 ng/ml of human RANKL (product no. 310-01, Peprotech, London, UK) was added to a proportion of the wells. Those wells with M-CSF and no RANKL were used as a negative control of osteoclastogenesis.
5
Isolation of CD14+ Monocytes from Human PBMCs
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Human PBMCs were obtained from healthy donors (Blood Bank, Umeå University Hospital) using the standard procedure of Lymphoprep centrifugation (07801, STEMCELL Technologies, Vancouver, British Columbia, Canada). The recovered cells were counted by Trypan Blue (15250-061, Gibco ThermoFisher Scientific) exclusion, revealing a routine viability of more than 99%. CD14-positive cells were isolated from PBMCs using the EasySep™ Human CD14 Positive Selection Kit II (17858, STEMCELL Technologies) following the manufacturer’s instructions. The Swedish Ethical Review authority approved the use of the donors’ blood, Ethical permit 2021-00913.
6
Monocyte-Derived Dendritic Cells and Macrophages
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Human monocytes were isolated from buffy coats using CD14 S-pluriBead antihuman beads (PluriSelect) or EasySep Human CD14 Positive Selection Kit II (Stemcell technologies) according to manufacturer’s instructions. The moDCs were generated by culturing monocytes in well plates for 5 d in RPMI1640 (Cytivia) medium supplemented with 10% (v/v) heat inactivated fetal calf serum (FCS; Thermo Fisher), 89 ng × mL−1 GM-CSF, and 22 ng × mL−1 IL-4 (both Immunotools). Medium was exchanged on day 3.
The monocyte-derived macrophages were generated by culturing monocytes in well plates in RPMI1640 (Cytivia) medium supplemented with 10% (v/v) heat inactivated FCS (Thermo Fisher) and 25 ng × mL−1 GM-CSF (Immunotools) for 7 d. Medium was exchanged on days 3 and 5.
The monocyte-derived macrophages were generated by culturing monocytes in well plates in RPMI1640 (Cytivia) medium supplemented with 10% (v/v) heat inactivated FCS (Thermo Fisher) and 25 ng × mL−1 GM-CSF (Immunotools) for 7 d. Medium was exchanged on days 3 and 5.
7
Generation of Monocyte-Derived Dendritic Cells
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The protocol for study of human blood was approved by the Institutional Review Board (approval number: 17-0075). PBMCs were purified from leukopack (NY Blood center) as described previously (14 (link)). To prepare MO-DCs, CD14+ monocytes were isolated from MO-DCs by EasySep Human CD14 positive selection kit II (StemCell Technologies) according to the manufacturer's protocol. CD14+ monocytes were cultured with RPMI1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% penicillin-streptomycin (P/S), 1% L-glutamine, 100 ng/ml of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) (PeproTech), and 50 ng/ml of recombinant human IL-4 (PeproTech) for 7 days. Cultures were kept at 37°C in a humidified atmosphere and 5% CO2. On day 7, MO-DCs were collected only from the non-adherent cells and the purity of MO-DCs was confirmed by flow cytometry with antibodies which were purchased from eBioscience (anti-HLA-DR-FITC: LN3 and anti-CD209-PE/Cy7: eB-h209) (23 (link)). Over 85% of HLA-DR+CD209+ MO-DCs were obtained consistently. We excluded adherent cells since cells shows mixed population with CD209+ and CD209- with various degrees (Figure S1A ).
8
In Vitro Phagocytosis Assay for Macrophages
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The in vitro phagocytosis assay was performed as described before24 (link),27 (link), with slight modifications. Buffy coats and human serum were obtained from the Swiss Blood Bank (Interregionale Blutspende SRK, Bern, Switzerland). PBMCs were enriched from buffy coats by density centrifugation using Lympho Spin Medium (pluriSelect). Monocytes were isolated from PBMCs using the EasySep Human CD14 Positive Selection Kit II (Stemcell Technologies) according to the manufacturer's instructions. 4–5 × 106 monocytes per well were differentiated into macrophages for 7 days in 6-well tissue culture plates in Iscove's modified Dulbecco's medium supplemented with 10% human serum, L-glutamine and penicillin/streptomycin. ACC-MESO-1 cells were harvested using non-enzymatic cell dissociation buffer (Sigma) and labelled with 5(6)-carboxyfluorescein diacetate N-succinimidyl ester (CFSE) in PBS at a final concentration of 20 µM. Macrophages were starved in serum-free medium for 2 h and 1 × 106 CFSE-labelled ACC-MESO-1 cells were added. Cell were co-cultured for 2 h in the presence of 10 µg/ml mouse anti-human CD47 (clone B6H12.2, ThermoFisher) or isotype control (mouse IgG1, ThermoFisher), then harvested, stained for CD45 and CD14 (Table S1) and analysed by FACS.
9
Isolation of CD14+ Monocytes from PBMCs
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Peripheral blood mononuclear cells [PBMCs] were isolated from buffy coats by density gradient centrifugation on Ficoll-Paque PREMIUM [GE Healthcare]. After washing in PBS and removal of erythrocytes as described above, CD14+ monocytes were isolated using CD14+ magnetic beads (EasySep Human CD14 Positive Selection Kit II [Stemcell Technologies, Canada]). Purity was evaluated on BD LSRFortessa X-20 and was > 90%.
10
Immuno-oncology Assay for T Cell-Mediated Cytokine Response
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Human T cells (5 × 104 per well) isolated from peripheral blood mononuclear cells (PBMCs) from a healthy donor using the EasySep Negative Human T Cell Kit (Cat# 19051, STEMCELL Technologies) were rested in R20 supplemented with 200 U/ml recombinant human IL-2 for 72 h. CD34+ blasts and CD14+ blasts were isolated from patients PBMC using the EasySep Human CD34 Positive Selection Kit II (Cat# 17856, STEMCELL Technologies) and the EasySep Human CD14 Positive Selection Kit II (Cat# 17858, STEMCELL Technologies), respectively. AML patients’ blasts from one CD34+ patient (myeloid), one CD14+ patient (monocytic), THP1 cells, and MOLM13 cells were co-cultured (5 × 104 per well) with the healthy donor’s T cells (5 × 104 per well) in R20 media in a U-bottom 96-well plate. Additionally, wells containing blasts alone, T cells alone, or a 1:1 ratio of T cells with Dynabeads Human T-Activator CD3/CD28 [Cat# 1161D, Thermo Fisher] were included for comparison. After 72 and 120 h of culture, the culture media were collected, and IFN-γ levels were assessed by ELISA using the LEGEND MAX™ Human IFN-γ ELISA Kit [Cat# 430107, BioLegend].
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