Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood samples by Lymphoprep (Cedarlane, Ontario, Canada) density gradient centrifugation. PBMCs were immediately used for cytotoxicity assay, and stored at −80°C in 10% dimethyl sulfoxide (DMSO; Sigma Aldrich, St. Louis, USA) and 90% fetal calf serum (FCS; Euroclone, Milan, Italy), for phenotypic and functional analyses. Cells were rapidly thawed at 37°C, washed, and rested overnight in RPMI 1640 medium supplemented with 10% FCS and 1% glutamine (all from Euroclone; complete medium), at 37°C in 5% CO2 atmosphere, prior to stimulation, immunostaining and cytofluorimetric analysis.
Lymphoprep
Manufactured by Cedarlane
Sourced in Canada
Lymphoprep is a density gradient medium used for the isolation of mononuclear cells from whole blood or other biological fluids. It separates the different blood cell populations based on their density.
Automatically generated - may contain errors
Lab products found in correlation
Glutamine, by Euroclone (1 mentions)
Fetal calf serum (fcs), by Euroclone (1 mentions)
Rpmi 1640 medium, by Euroclone (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Sodium citrate, by Merck Group (1 mentions)
Recombinant human il 4, by Miltenyi Biotec (1 mentions)
Recombinant human gm csf, by Miltenyi Biotec (1 mentions)
Fetal bovine serum (fbs), by Euroclone (1 mentions)
Penicillin streptomycin, by Lonza (1 mentions)
Rpmi 1640 medium, by Lonza (1 mentions)
Cd14 microbead, by Miltenyi Biotec (1 mentions)
L glutamine, by Lonza (1 mentions)
Adenosine triphosphate (atp), by Merck Group (1 mentions)
5 protocols using lymphoprep
1
PBMC Isolation and Cryopreservation
2
PBMC Isolation from Buffy Coats
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Blood was drawn from donors attending the Transfusion center of Pisa University Hospital or from healthy volunteers after an informed consent was obtained. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the local Ethical Committee (Comitato Etico Area Vasta Nord-Ovest, CEAVNO, Protocol 34743, 28 June 2018). PBMCs were isolated from buffy coats by standard gradient separation as described previously [21 (link)]. Briefly, buffy coats were diluted 1:1 with PBS, 10% sodium citrate (v/v) (Merck). Cell suspensions were layered on a density gradient (Lymphoprep, Cedarlane, ON, Canada), and subjected to 20 min centrifugation at 160× g at room temperature. Afterwards, platelets in the supernatant were gently removed without disturbing the mononuclear layer at the interface. After a further centrifugation at 800× g for 20 min, PBMCs were collected from the interface and washed three times in PRMI. Finally, PBMCs were resuspended in complete RPMI, replacing the fetal bovine serum with 10% heat-inactivated autologous plasma.
3
Isolation and Differentiation of Dendritic 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 healthy peripheral blood by density gradient centrifugation over Lymphoprep (CEDARLANE) and washed with PBS. Red blood cells were lysed by incubation with ammonium chloride solution and platelets were removed by low-speed centrifugation.
CD14+ monocytes were isolated from PBMCs by positive selection using CD14 MicroBeads (Miltenyi Biotec) according to manufacturer’s instructions.
CD14+ monocytes were differentiated into dendritic cells (DC) in RPMI 1640 medium (Lonza), 10% fetal bovine serum (FBS) (Euroclone), 100 U/mL penicillin/streptomycin (Lonza), and 2 mM L-glutamine (Lonza) at 106 cells/mL, in the presence of 100 ng/mL Recombinant Human GM-CSF (Miltenyi Biotec) and 10 ng/mL Recombinant Human IL-4 (Miltenyi Biotec) for 7 days at 37°C with 5% CO2. Fresh medium with cytokines, at concentrations stated above, was added on day 3. Mature DC were obtained by adding 1 mg/mL lipopolysaccharide at day 5 of differentiation.
DC-10 were generated as previously described.17 (link) Briefly, CD14+ monocytes were cultured as described above for DC differentiation, with the addition of Recombinant Human IL-10 (CellGenix) at 10 ng/mL.
CD14+ monocytes were isolated from PBMCs by positive selection using CD14 MicroBeads (Miltenyi Biotec) according to manufacturer’s instructions.
CD14+ monocytes were differentiated into dendritic cells (DC) in RPMI 1640 medium (Lonza), 10% fetal bovine serum (FBS) (Euroclone), 100 U/mL penicillin/streptomycin (Lonza), and 2 mM L-glutamine (Lonza) at 106 cells/mL, in the presence of 100 ng/mL Recombinant Human GM-CSF (Miltenyi Biotec) and 10 ng/mL Recombinant Human IL-4 (Miltenyi Biotec) for 7 days at 37°C with 5% CO2. Fresh medium with cytokines, at concentrations stated above, was added on day 3. Mature DC were obtained by adding 1 mg/mL lipopolysaccharide at day 5 of differentiation.
DC-10 were generated as previously described.17 (link) Briefly, CD14+ monocytes were cultured as described above for DC differentiation, with the addition of Recombinant Human IL-10 (CellGenix) at 10 ng/mL.
4
Isolation of PBMCs from Buffy Coats
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Blood was drawn from donors attending the Transfusion center of Pisa University Hospital or from healthy volunteers after an informed consent was obtained. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the local Ethical Committee (Comitato Etico Area Vasta Nord-Ovest, CEAVNO, Protocol 34743, 28 June 2018).
PBMC were isolated from buffy coats by standard gradient separation as described previously [15 (link)]. Briefly, buffy coats were diluted 1:1 with PBS, 10% sodium citrate (v/v). Cell suspensions were layered on a density gradient (Lymphoprep, Cedarlane, ON, Canada), and subjected to 20 min centrifugation at 160× g at RT. Afterward, platelets in the supernatant were gently removed without disturbing the mononuclear layer at the interface. After a further centrifugation at 800× g for 20 min, PBMC were collected from the interface and washed three times in PRMI. Finally, PBMC were resuspended in complete RPMI, replacing the fetal bovine serum with 10% heat-inactivated autologous plasma.
PBMC were isolated from buffy coats by standard gradient separation as described previously [15 (link)]. Briefly, buffy coats were diluted 1:1 with PBS, 10% sodium citrate (v/v). Cell suspensions were layered on a density gradient (Lymphoprep, Cedarlane, ON, Canada), and subjected to 20 min centrifugation at 160× g at RT. Afterward, platelets in the supernatant were gently removed without disturbing the mononuclear layer at the interface. After a further centrifugation at 800× g for 20 min, PBMC were collected from the interface and washed three times in PRMI. Finally, PBMC were resuspended in complete RPMI, replacing the fetal bovine serum with 10% heat-inactivated autologous plasma.
5
Monocyte Inflammasome Activation by ATP
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Venous blood was collected in EDTA tubes and immediately centrifuged at 1,400 rpm for 15 minutes to remove plasma. Peripheral blood mononuclear cells were isolated using Lymphoprep (Cedarlane, Ontario, Canada) according to manufacturer's instructions and as previously described (20) . Cells were re-suspended in Dulbecco's Modified Eagle Medium supplemented with 5 % fetal bovine serum and a full blood cell count was performed.
Monocytes were seeded at 8x10 4 cells/well on a 48-well plate in growth media and nonadherent cells were removed 1 hour later. Cells were cultured for 3 hours, followed by stimulation of half the cells with ATP (Sigma-Aldrich, St Louis, USA; 5 mM) for 30 minutes (Figure 1). Notably, ATP stimulation was conducted to activate the P2X7 receptor leading to potassium efflux, thereby completing inflammasome activation (8) . Monocyte lysates and cell media were collected and stored at -80°C for downstream analysis.
Monocytes were seeded at 8x10 4 cells/well on a 48-well plate in growth media and nonadherent cells were removed 1 hour later. Cells were cultured for 3 hours, followed by stimulation of half the cells with ATP (Sigma-Aldrich, St Louis, USA; 5 mM) for 30 minutes (Figure 1). Notably, ATP stimulation was conducted to activate the P2X7 receptor leading to potassium efflux, thereby completing inflammasome activation (8) . Monocyte lysates and cell media were collected and stored at -80°C for downstream analysis.
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!