Peripheral blood mononuclear cells (PBMCs) were separated by centrifugation in lymphoprepTM density gradient medium (#07861, StemCell Technologies, Vancouver, BC, Canada) from buffy coat leukocyte concentrates obtained from healthy female donors provided by the Hong Kong Red Cross. CD8+ T cells were purified by incubating the PBMCs with RosetteSep human CD8+ T cell Enrichment Cocktail (#15063, StemCell Technologies) before gradient centrifugation according to the instructions recommended by the supplier. For CD8+ T cell culture, complete RPMI medium containing 10% FBS and 100 U/mL penicillin-streptomycin was used.
Lymphoprep density gradient medium
Manufactured by STEMCELL
Sourced in Canada, United Kingdom, United States, Germany
Lymphoprep is a density gradient medium used to isolate mononuclear cells, such as lymphocytes and monocytes, from whole blood or other cell suspensions. It allows for the separation of these cells based on their density differences when centrifuged.
Automatically generated - may contain errors
Lab products found in correlation
Sepmate tube, by STEMCELL (8 mentions)
Vacutainer k2 edta tubes, by BD (4 mentions)
Sepmate 50 tube, by STEMCELL (4 mentions)
Easysep human t cell isolation kit, by STEMCELL (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
L glutamine, by Thermo Fisher Scientific (3 mentions)
Trypan blue, by Merck Group (2 mentions)
Easysep human monocyte isolation kit, by STEMCELL (2 mentions)
Hepes, by Thermo Fisher Scientific (2 mentions)
Nucleocounter nc 3000, by ChemoMetec (2 mentions)
Rosettesep human b cell enrichment cocktail, by STEMCELL (2 mentions)
Mem medium, by Thermo Fisher Scientific (2 mentions)
Sodium heparin anticoagulant collection tubes, by BD (2 mentions)
Alsever s solution, by Merck Group (2 mentions)
Trypsin edta, by Thermo Fisher Scientific (2 mentions)
Sepmate pbmc isolation tube, by STEMCELL (2 mentions)
Rosettesep human cd8 t cell enrichment cocktail, by STEMCELL (1 mentions)
Vacutainer acd tubes, by BD (1 mentions)
Biotinylated anti epcam antibody, by R&D Systems (1 mentions)
Human cd14 microbeads, by Miltenyi Biotec (1 mentions)
69 protocols using lymphoprep density gradient medium
1
Isolation and Culture of CD8+ T Cells
2
Enrichment of Circulating Tumor Cells from Peripheral Blood
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For sample collection, 18 mL of peripheral blood was obtained from the patients. The first 2 mL was collected in BD vacutainer K2 EDTA tubes, and the remaining 16 mL was collected in two BD vacutainer ACD tubes (8 mL per tube). Whole blood samples were purified using LymphoprepTM density gradient medium (STEMCELL Technologies, Vancouver, BC, Canada) for the enrichment of the peripheral blood mononuclear cell (PBMC) fraction.
Isolated PBMCs were fixed with 4% paraformaldehyde for 15 min at room temperature. Fixed PBMCs were then treated with an antibody cocktail containing biotinylated anti-EpCAM antibody (R&D Systems, Minneapolis, MN, USA) and biotinylated anti-E-cadherin antibody (R&D Systems, Minneapolis, MN, USA) and mixed consistently for 30 min at 37 °C. Then, 3 mL of Dulbecco’s phosphate-buffered saline was added to the mixture, which was centrifuged at 400× g for 5 min to collect the cell pellets and remove the supernatant.
Isolated PBMCs were fixed with 4% paraformaldehyde for 15 min at room temperature. Fixed PBMCs were then treated with an antibody cocktail containing biotinylated anti-EpCAM antibody (R&D Systems, Minneapolis, MN, USA) and biotinylated anti-E-cadherin antibody (R&D Systems, Minneapolis, MN, USA) and mixed consistently for 30 min at 37 °C. Then, 3 mL of Dulbecco’s phosphate-buffered saline was added to the mixture, which was centrifuged at 400× g for 5 min to collect the cell pellets and remove the supernatant.
3
Isolation and Culture of Human Monocytes
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Buffy coats blood was obtained from Hong Kong Red Cross Blood Transfusion Service. CD14+ monocytes were isolated by positive selection method with human CD14 MicroBeads (Miltenyi Biotec, Germany) as we described previously31 (link). Briefly, peripheral blood mononuclear cells (PBMCs) were first isolated by gradient centrifugation in LymphoprepTM density gradient medium (STEMCELL Technologies, Vancouver, Canada). PBMCs were then incubated with CD14 microbeads for 15 min at 4 °C. The cells were washed once and loaded onto a MACS column placed in the magnetic field. The column was washed three times before the elution of the labelled CD14+ monocytes. The purity of the obtained monocytes was confirmed to be more than 95% by flow cytometry (data not shown). The monocytes were cultured in RPMI-1640 complete medium supplemented with GM-CSF (10 ng/ml) and IL4 (10 ng/ml), 10% FBS, 1% penicillin-streptomycin, 1% GlutaMAX, 1 mM sodium pyruvate, 1% non-essential amino acid, and 50 μM 2-mercaptoethanol for the experiments except that FBS was left out during the one-hour viral absorption. Protocol for using buffy coats blood from healthy blood donors was approved by the Institutional Review Board of the University of Hong Kong (ref no. IRB UW16-106).
4
Isolation of Peripheral Blood Mononuclear Cells
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Femoral blood was collected in K2-EDTA vacutainer tubes (BD, 367841). Within four hours of collection, blood was centrifuged at 1200rpm for 20 minutes to separate the plasma. The remaining blood cells were layered over a Lymphoprep TM Density Gradient Medium from STEMCELL Technologies. Then, PBMCs were isolated by density gradient centrifugal separation (54) (link).
5
Isolation and differentiation of human immune cells
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Human peripheral blood mononuclear cells (PBMCs) were isolated from fresh whole blood by Lymphoprep density gradient medium in SepMate tubes (Stemcell) and cell number was enumerated by 0.4% (v/v) trypan blue (Sigma) on a haemocytometer. PBMCs were used for CD14+ CD16- monocyte isolation using EasySep Human Monocyte Isolation Kit (Stemcell). Freshly isolated monocytes were then differentiated into mature or immature dendritic cells (DCs) using ImmunoCult DC Culture Kit according to the manufacturer’s instructions (Stemcell). Thawed PBMCs were treated with 100μg/mL DNAse I solution (Stemcell) for 15 minutes at room temperature before downstream use. CD3+ T cells were isolated from PBMCs using EasySep Human T Cell Isolation Kit (Stemcell).
6
Activation and Transduction of Primary Human CD4+ T Cells
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Human primary CD4+ T cells were obtained from peripheral blood mononuclear cells (PBMCs) from healthy volunteer donors through the Infectious Diseases BioBank at King’s College London (ethics reference MM2-220518) under overall permission from the Southampton and South West Hampshire Research Ethics Committee (REC; B) (REC reference 19/SC/0232). PBMCs were isolated using density gradient centrifugation in SepMate tubes (STEMCELL Technologies) with Lymphoprep density gradient medium (STEMCELL Technologies). Total CD4+ T cells were isolated using a Human CD4+ T Cell Isolation Kit (Miltenyi Biotec). CD4+ T cells were cultured in RPMI-1640 medium with GlutaMAX and HEPES supplemented with 10% heat-inactivated autologous human serum and 1% penicillin-streptomycin (Life Technologies). Cells were then activated using Dynabeads Human T-Activator CD3/CD28 (Life Technologies) and recombinant human interleukin-2 (30 U/mL; Roche) for 48 h prior to infection.
Activated CD4+ T cells were transduced with lentiviral vectors (1−10 ng of p24Gag of vector per 50,000 cells). 48 h post-transduction, the cells were fixed in 4% paraformaldehyde in DPBS before assessing transduction efficiency by flow cytometry measuring intracellular GFP expression.
Activated CD4+ T cells were transduced with lentiviral vectors (1−10 ng of p24Gag of vector per 50,000 cells). 48 h post-transduction, the cells were fixed in 4% paraformaldehyde in DPBS before assessing transduction efficiency by flow cytometry measuring intracellular GFP expression.
7
Monocyte and Macrophage Cell Culture Protocol
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THP-1 monocytes (ATCC TIB-202) and human peripheral blood mononuclear cells (PBMCs) were maintained in RPMI 1640 medium (Gibco, Gaithersburg, MD). RPMI 1640 was supplemented with 2 mM l -glutamine, 100 IU/ml penicillin, 100 μg/ml streptomycin, 10 mM HEPES, and 10% heat-inactivated fetal bovine serum purchased from Gibco. THP–PPM1A+ and THP–△PPM1A cells were generated previously as described (Sun et al. 2016 (link), Berton et al. 2022 (link)). Cells were maintained at 37°C in a humidified atmosphere of 5% CO2. THP-1 monocytes were differentiated with 100 ng/ml phorbol ester 13-phorbol-12-myristate acetate (PMA, Alfa Aesar, Haverhill, MA) for 72 h. PBMCs were collected according to approved ethics protocol (#2005388-01H) and isolated via density centrifugation method using the Lymphoprep density gradient medium (StemCell Technologies). Positive selection of monocytes was performed using anti-CD14 coated magnetic particles from StemCell Technologies according to the manufacturer’s protocol. Monocytes were differentiated with 5 ng/ml GM-CSF (Gibco) for 9 days to obtain hMDMs.
8
T Cell Isolation and Activation Protocol
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Peripheral blood mononuclear cells (PBMC) were isolated from fresh leukopaks using Lymphoprep density gradient medium (STEMCELL Technologies, Vancouver, Canada) and cryopreserved using standard methods. Upon thaw, T cells were isolated from PBMC using soluble CD3 (clone OKT3) and CD28 (clone 15E8) antibodies (Miltenyi Biotec, Bergisch Gladbach, Germany), each at 100 ng/mL. Cells were initiated for 3 days in complete culture medium consisting of CTS OpTmizer plus supplement (Gibco, Waltham, MA, USA) with 5% Physiologix serum replacement (Nucleus Biologics, San Diego, CA, USA), 1% L-glutamine and 250 IU/mL IL-2 (CellGenix, Freiburg, Germany). Cell viability was measured using a 4′,6-diamidino-2-phenylindole/Acridine Orange viability stain with the Via1-Cassette and NucleoCounter NC-3000 (ChemoMetec, Allerod, Denmark).
9
Isolation of PBMCs from Blood
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PBMCs were isolated from the blood of healthy donors and patients with PDAC using Lymphoprep™ density gradient medium in Sepmate™-50 tubes (StemCell Technologies). Isolated PBMCs were immediately frozen in FBS with 10% DMSO until further use. Samples were obtained under the approval of the Oxford Radcliffe Biobank research tissue bank ethics and after informed, written consent of the patients.
10
Isolation and Activation of Immune Cells
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Blood was recruited from healthy donors at St. Olavs Hospital HF, the Bloodbank (project approved by Regional Ethical Committee of Mid-Norway; #2016/553). Peripheral blood mononuclear cells (PBMC) were isolated using SepMate separation tubes with LymphoPrep density gradient medium from STEMCELL Technologies (Cambridge, UK), according to the manufacturer’s recommendations. For experiments, 1 x 106 cells per well were plated in 1 mL Roswell Park Memorial Insitute (RPMI) medium supplemented with 5% fetal bovine serum (FBS), 0.3 mg/mL glutamine, and 0.1 mg/mL gentamicin. Inhibitors were added 2 h prior to the addition of the Ca++ ionophore A23178 (30 µM, 15 min) to activate cPLA2α or lipopolysaccharide (LPS) (10 ng/mL, 72 h) as a potent inducer of inflammation. Following treatment, the cell suspensions were centrifuged to isolate the supernatant from the cell fraction. Samples were stored at −80 °C until analysis.
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