Primary samples from patients suffering from MCL and CLL were collected at the time of diagnosis with informed consent from San Gerardo Hospital (HSG, Monza, Italy), according to the IRB‐approved protocol (HSG IRB #926, code: CD24.DEM) accepted by Local Ethical Committee. Tumour population was obtained from patients' blood that was diluted with PBS (dilution 1:2) and separated on Lympholyte‐H from Cedarlane to get PBMCs. Tumour cells were then isolated from PBMCs by CD19 Microbeads isolation kit from Miltenyi Biotec according to the manufacturer's protocol. Isolated cancer cells were then checked for purity by flow cytometry (CD45+/CD19+ cells >90% by flow cytometry). Patients‐derived monocytes were selected with the same protocol used for donors' monocytes. Leftover samples from healthy donors for PBMCs and monocytes collection were obtained after plateletpheresis in Transfusion Medicine Unit (HSG, Monza, Italy), after signing informed consent. All the procedures outlined in the study were conducted in accordance with the ethical principles of Helsinki Declaration.
Lympholyte h
Manufactured by Cedarlane
Sourced in Canada, United States, Netherlands, Italy
Lympholyte-H is a density gradient medium used for the isolation and purification of lymphocytes from whole blood or other biological samples. It is designed to separate mononuclear cells, such as lymphocytes, from other cellular components through centrifugation.
Automatically generated - may contain errors
Lab products found in correlation
Dynabeads human t activator cd3 cd28 beads, by Thermo Fisher Scientific (7 mentions)
Dynabeads untouched human cd4 t cells kit, by Thermo Fisher Scientific (7 mentions)
Facscanto 2, by BD (6 mentions)
Recombinant human il 2, by Thermo Fisher Scientific (6 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Streptomycin, by Thermo Fisher Scientific (4 mentions)
Penicillin, by Thermo Fisher Scientific (4 mentions)
Rpmi 1640, by Thermo Fisher Scientific (3 mentions)
Ls column, by Miltenyi Biotec (3 mentions)
Quadromacs separator, by Miltenyi Biotec (3 mentions)
Anti pe microbeads, by Miltenyi Biotec (3 mentions)
L glutamine, by Lonza (3 mentions)
Hm csf, by Thermo Fisher Scientific (3 mentions)
Collagenase, by Merck Group (3 mentions)
100 μm strainer, by BD (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Pan monocyte isolation kit, by Miltenyi Biotec (3 mentions)
Countbright absolute counting beads, by Thermo Fisher Scientific (2 mentions)
Interleukin 4 (il 4), by R&D Systems (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
121 protocols using lympholyte h
1
Isolation of Primary Tumor Cells
2
Plasmid-mediated Silencing of PKD2
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Culture media, saline buffers and plastic material were obtained from EuroClone (Milan, Italy). Anti-P-mTOR, anti-mTOR, anti-P-ERK, anti-ERK, and anti-MIF antibodies were purchased from Cell Signaling Technology (EuroClone). Anti PC1 7E12, anti-β-actin and anti-NFkB antibodies were bought from Santa Cruz Biotechnology (Milan, Italy). Anti-CD3 (OKT3) monoclonal antibody was purchased from Invitrogen (Thermo Fisher Scientific, Milan, Italy). The anti-PC2 N-ter antibody was kindly provided by Prof. Stefan Somlo (Yale University, CT, USA). Enhanced chemiluminescent substrates for Western blotting, HRP-conjugated goat anti-rabbit and anti-mouse antibodies were purchased from Cell Signaling Technology (EuroClone). Lympholyte®-H and phytohaemagglutinin (PHA) were obtained from Cedarlane Laboratories (Tebu-Bio, Milan, Italy) and EuroClone, respectively. Interleukin-2 (IL-2) and Fura 2-AM were purchased from Invitrogen. Platelet-activating factor (PAF), ATP and ionomycin were purchased from Sigma-Aldrich (Milan, Italy). The recombinant plasmid for PKD2 silencing (TRPP2-siRNA) was constructed in our laboratory by using the pSuper vector (Oligoengine, Seattle, WA) as previously described [12 (link)].
3
PBMC Isolation from Peripheral Blood
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Fresh peripheral blood samples were collected into EDTA-K2-containing tubes and centrifuged at 800 × g for 5 min. Then, the plasma was collected into the tubes and stored at -80 °C. The remaining blood components were diluted with an equal volume of phosphate buffered saline (PBS), and then PBMCs were collected by gradient centrifugation according to the Lympholyte®-H (CEDARLANE, Netherlands) manufacturer’s protocols. The total RNA in PBMCs from each case was extracted using an RNA Extraction Kit (Vazyme Biotech, Nanjing, China) as quickly as possible according to the protocol. The integrity and concentration of the RNA were assessed by a NanoDrop 2000 (Thermo Scientific, USA) spectrophotometer.
4
Blood Isolation and PBMC Extraction
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For blood isolation, mice (7 weeks of age) were terminally anesthetized with ketamine-xylazine based on the weight of the animal, then the posterior vena cava was severed, and blood collected by heparinized capillary (Fisherbrand) to BD Microtainer blood collection tubes with K2EDTA (BD) and additional tissues were isolated for RNA extraction. All relevant ethical regulations for work with human participants were complied with and patient blood samples were collected following written informed consent as approved by the University of Florida Institutional Review Board (IRB). Sampling age is included in Supplementary Data 1 . Blood was collected into DNA/RNA Shield Blood Collection Tubes (Zymo Research) or BD Vacutainer Tubes, ACD A (BD Biosciences). Peripheral blood mononuclear cells (PBMC) were isolated from the buffy coat of collected whole blood, followed by density gradient separation using Lympholyte-H according to manufacturer’s provided protocol (Cedarlane). Red blood cells were lysed and removed by using the RBC Lysis Buffer (Roche). PBMCs were washed with PBS and used for either genomic DNA isolation (Flexigene kit; Qiagen), total RNA extraction (TRIzol).
5
Isolation and Culture of PBMCs and HEK-CCR2b Cells
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Peripheral blood mononuclear cells (PBMC) were isolated from whole blood taken from healthy volunteers. Ethical approval to obtain blood from healthy volunteers was granted by the Research Ethics Committee (12/NE/0121). PBMC were isolated from heparinised blood using Lympholyte-H (Cedarlane Laboratories) as per manufacturer’s instructions. Cells were serum starved for 1 hr prior to use in assays.
HEK-CCR2b cells40 (link) were cultured in complete DMEM with 800 μg/ml G418 (Calbiochem). The THP-1 monocytic cell line (ATCC TIB-202) was cultured in complete RPMI-1640.
HEK-CCR2b cells40 (link) were cultured in complete DMEM with 800 μg/ml G418 (Calbiochem). The THP-1 monocytic cell line (ATCC TIB-202) was cultured in complete RPMI-1640.
6
PBMC Isolation and Protein Analysis
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Peripheral blood mononuclear cells (PBMCs) from 20 mL of ethylenediamine-tetra acetic acid (EDTA) anticoagulated blood were isolated by Lympholyte-H (Cedarlane, BVT, Canada). p22phox protein expression and MYPT-1 phosphorylation state were assessed by Western blot analysis as previously reported [8 (link),9 (link)].
7
Isolation and Stimulation of PBMCs
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PBMCs were isolated by Ficoll-Hypaque density centrifugation (Lympholyte-H; Cedarlane Laboratories, Hornby, Ontario, Canada). Whole blood was layered on top of Ficoll and centrifuged at 800 g for 40 min. After centrifugation, the interphase containing PBMCs was collected and washed twice in phosphate-buffered saline (PBS) (GIBCO; Invitrogen Laboratories, UK). Then cells were counted, and viability was determined by trypan blue exclusion test. After that, cells were resuspended in phenol red free RPMI 1640 medium (GIBCO; Invitrogen Laboratories, UK), 5% charcoal-dextran-stripped fetal bovine serum (FBS), and 1% penicillin/streptomycin solution (GIBCO; Invitrogen Laboratories, UK) at a final concentration of 2 × 106 cells/ml in 12-well flat-bottom culture plates. After 4 hr incubation at 37 °C in 5% CO2 humidified atmosphere, the cultures were treated with concentrations of 1×10-6 M and 1×10-8 M 17β-estradiol (Sigma-Aldrich) for 24 hr. We used a dose of 1×10-8 M 17β-estradiol to treat PBMCs, because this value falls near the mean±SEM concentration of estrogen detected in human serum at the maximum preovulatory (i.e., 2,393±356 pmol/l)(23 (link)). After incubation, PBMCs were collected in order to evaluate mRNA expression and iNOS activity. In addition, the cell-free supernatants were collected and stored at -70 °C until assay.
8
Multiparametric Flow Cytometry Analysis
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Mononuclear cells were isolated from the peripheral blood by Ficoll gradient centrifugation (Lympholyte-H, Cedarlane), and flow cytometry analysis, using the BD LSRFortessa flow cytometer and FACSDiva 7 Software, was performed in 4 tubes using the following conjugated antibodies (Becton Dickinson, Franklin Lakes, NJ): anti-CD3 (V500), anti-CD4 (allophycocyanin [APC]-H7), anti-CD8 (PercCP-Cy5.5), anti-CD28 (phycoerythrin [PE], anti-CD25 (PE.Cy7), anti-CD127 (BV421), anti-CD45RA (APC), anti-CCR6 (APC), anti-CD161 (B421), anti-CCR4 (PE.Cy7), and anti-CXCR3 (PE) for effector (Teff) and regulatory T (Treg) cells; anti-CD19 (PE.Cy7), anti-CD38 (APC), and anti-CD24 (PE) for B cells; and anti-CD3 (V500), anti-CD16 (PerCp-Cy5.5), and anti-CD56 (BV421) for natural killer (NK) cells.
Flow-Count Fluorospheres (Beckman Coulter, Galway, Ireland) were used to obtain absolute cell counts.
Flow-Count Fluorospheres (Beckman Coulter, Galway, Ireland) were used to obtain absolute cell counts.
9
Isolation of T Cells from Healthy Donors
10
Activation and Purification of Human T Cells
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Peripheral blood mononuclear cells (PBMCs) (STEMCELL Technologies, Vancouver, Canada) from male healthy donors (25–32 years old) were cultured at 2.25 × 106 cells/ml in complete T cell media: RPMI 1640 (ATCC 30–2001), 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 4500 mg/L glucose, and 1500 mg/L sodium bicarbonate, 1x antibiotic-antimycotic (Gibco), 1x non-essential amino acids (Gibco) and 10 ng/ml recombinant human IL-2 (R&D Systems, Minneapolis, MN). Products were obtained using Institutional Review Board (IRB) approved consent forms and protocols. Cells were stimulated with α-CD3/CD28 Dynabeads (Gibco) for 6 days at a 1:1 ratio of cells:beads unless otherwise noted. After 6 days, viable cells were purified using Lympholyte®-H (Cedar Lane, Burlington, NC). Cells were restimulated for 6 h with α-CD3/CD28 Dynabeads.
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