PBMCs were isolated from 20 mL of heparinized blood by density gradient centrifugation using Pancoll human solution (PAN-Biotech GmbH, Aidenbach, Germany). First, the blood was diluted 1.5× with phosphate-buffered saline (PBS). This mixture was gently pipetted onto the Pancoll solution. The gradient was immediately centrifuged for 30 min with 1000× g without breaks. The plasma was discarded, and the mononuclear cells layer was transferred into a new tube, washed twice with PBS and finally once with AIM-V medium 10 min at 700× g. Then, cells were counted and adjusted with AIM-V medium to a concentration of 2 × 106 cells per ml.
Pancoll human solution
Manufactured by PAN Biotech
Sourced in Germany
Pancoll human solution is a sterile, endotoxin-tested, cell culture medium used for the isolation and separation of human mononuclear cells from whole blood or buffy coat. It is a density gradient medium designed to efficiently isolate viable and functional mononuclear cells.
Automatically generated - may contain errors
Lab products found in correlation
Human pan t cell isolation kit, by Miltenyi Biotec (2 mentions)
Rat tail collagen type 1, by BD (2 mentions)
Anti biotin microbeads staining, by Miltenyi Biotec (2 mentions)
Anti cd25 biotin conjugated antibody, by Miltenyi Biotec (2 mentions)
Egm 2mv medium, by Lonza (2 mentions)
1 2 13c glucose, by Cambridge Isotopes (1 mentions)
Ampuwa, by Fresenius (1 mentions)
Pig serum, by Bio-Rad (1 mentions)
U 13c glucose, by Cambridge Isotopes (1 mentions)
4 5 6 13c glucose, by Cambridge Isotopes (1 mentions)
Aim 5 medium, by Thermo Fisher Scientific (1 mentions)
5 protocols using pancoll human solution
1
PBMC Isolation from Heparinized Blood
2
Isotopic Glucose-Labeling Metabolomics
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RPMI 1640 powder (without glutamine, glucose, and NaHCO3) was produced by Genaxxon bioscience (Ulm, Germany), and the derivatization reagents N,O-bis(trimethylsilyl)-trifluoroacetamide (BSTFA) were purchased from abcr (Karlsruhe, Germany). Pig serum was obtained from Bio-Rad Laboratories (Hercules, CA, USA) and Pancoll human solution (density of 1.119 g/mL and density of 1.077 g/mL, respectively) were purchased from PAN Biotech (Aidenbach, Germany). The pHrodo™ Green E. coli BioParticles™ phagocytosis kit for flow cytometry and phosphate buffer saline (PBS, without Ca2+, Mg2+) were produced by Thermo Fisher Scientific (Waltham, MA, USA). We obtained Ampuwa (aqua ad iniectabilia) and sterile 0.9% NaCl solution from Fresenius Kabi (Bad Homburg, Germany). [1,2-13C]glucose (99 atom% 13C), [4,5,6-13C]glucose (99.5%), and [U-13C]glucose (99%), [U-13C]glucose-D-6-phosphate disodium salt hydrate (99%) were purchased from Cambridge Isotope Laboratories (Andover, MA, USA). All other chemicals and standard substances were purchased from Sigma-Aldrich (St. Louis, MO, USA).
3
Isolation and Co-culture of ECFCs and T Cells
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CB samples from healthy full term newborns were obtained from the CB Bank of St Louis Hospital (Paris, France). Human APB from healthy adults was obtained from the French Establishment of Blood (EFS) (Rungis, France). Mononuclear cells (MNCs) were obtained by density gradient centrifugation using Pancoll human solution (Pan-Biotech).
For ECFC isolation, MNCs were seeded into rat-tail collagen type-I (BD-Bioscience) coated wells as previously described [26 (link)]. ECFC colonies appeared after 7–20 days of culture. From passage 1 (P1), cells were seeded at 5000 cells/cm2 and grew in EGM-2MV medium (Lonza).
Human pan T cell isolation kit (Miltenyi-Biotec) was used to isolate total CD3+ T cells from MNCs of peripheral adult blood. Furthermore, CD25+ cells were depleted from the CD3+ T cell population using anti-CD25 biotin conjugated antibody (Miltenyi-Biotec), followed by anti-biotin microbeads staining (Miltenyi-Biotec). Then, the magnetic-activated cell sorting (MACS) method was used in all cell isolations. The resulting CD3+CD25− T cells, more than 93% purity, were cultured in the presence of ECFCs. The isolation of T cells from co-culture in presence of ECs is based on the biological capacity of ECs to adhere to plastic plates, and T cells that stay in suspension; hence, they were collected with gentle resuspension and aspiration.
For ECFC isolation, MNCs were seeded into rat-tail collagen type-I (BD-Bioscience) coated wells as previously described [26 (link)]. ECFC colonies appeared after 7–20 days of culture. From passage 1 (P1), cells were seeded at 5000 cells/cm2 and grew in EGM-2MV medium (Lonza).
Human pan T cell isolation kit (Miltenyi-Biotec) was used to isolate total CD3+ T cells from MNCs of peripheral adult blood. Furthermore, CD25+ cells were depleted from the CD3+ T cell population using anti-CD25 biotin conjugated antibody (Miltenyi-Biotec), followed by anti-biotin microbeads staining (Miltenyi-Biotec). Then, the magnetic-activated cell sorting (MACS) method was used in all cell isolations. The resulting CD3+CD25− T cells, more than 93% purity, were cultured in the presence of ECFCs. The isolation of T cells from co-culture in presence of ECs is based on the biological capacity of ECs to adhere to plastic plates, and T cells that stay in suspension; hence, they were collected with gentle resuspension and aspiration.
4
Isolation and Enumeration of PBMCs
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PBMCs were freshly isolated from 20 mL heparinized blood by density gradient centrifugation using Pancoll human solution (PAN-Biotech GmbH, Aidenbach, Germany). First, the blood was diluted to half volume with phosphate-buffered saline (PBS). This mixture was pipetted gently onto the Pancoll solution using half the volume of the PBS-diluted blood solution. The gradient was immediately centrifuged for 30 min with 100× g without a break. The plasma was discarded and the mononuclear cell layer was transferred into a new 50 mL tube, washed twice with PBS and finally once with AIM-V medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) for 10 min at 700× g, respectively. Then, cells were counted and adjusted to a concentration of 2 × 106 cells/mL in AIM-V medium.
5
Isolation and Co-culture of ECFCs and T Cells
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CB samples from healthy full term newborns were obtained from the CB Bank of St Louis Hospital (Paris, France). Human APB from healthy adults was obtained from the French Establishment of Blood (EFS) (Rungis, France). Mononuclear cells (MNCs) were obtained by density gradient centrifugation using Pancoll human solution (Pan-Biotech).
For ECFC isolation, MNCs were seeded into rat-tail collagen type-I (BD-Bioscience) coated wells as previously described. 26 ECFC colonies appeared after 7-20 days of culture. From passage 1 (P1), cells were seeded at 5000 cells/cm 2 and grew in EGM-2MV medium (Lonza).
Human pan T cell isolation kit (Miltenyi-Biotec) was used to isolate total CD3 + T cells from MNCs of peripheral adult blood. Furthermore, CD25 + cells were depleted from the CD3 + T cell population using anti-CD25 biotin conjugated antibody (Miltenyi-Biotec), followed by anti-biotin microbeads staining (Miltenyi-Biotec). Then, the magnetic-activated cell sorting (MACS) method was used in all cell isolations.
The resulting CD3 + CD25 -T cells, more than 93% purity, were cultured in the presence of ECFCs. The isolation of T cells from co-culture in presence of ECs is based on the biological capacity of ECs to adhere to plastic plates, and T cells that stay in suspension; hence, they were collected with gentle resuspension and aspiration.
For ECFC isolation, MNCs were seeded into rat-tail collagen type-I (BD-Bioscience) coated wells as previously described. 26 ECFC colonies appeared after 7-20 days of culture. From passage 1 (P1), cells were seeded at 5000 cells/cm 2 and grew in EGM-2MV medium (Lonza).
Human pan T cell isolation kit (Miltenyi-Biotec) was used to isolate total CD3 + T cells from MNCs of peripheral adult blood. Furthermore, CD25 + cells were depleted from the CD3 + T cell population using anti-CD25 biotin conjugated antibody (Miltenyi-Biotec), followed by anti-biotin microbeads staining (Miltenyi-Biotec). Then, the magnetic-activated cell sorting (MACS) method was used in all cell isolations.
The resulting CD3 + CD25 -T cells, more than 93% purity, were cultured in the presence of ECFCs. The isolation of T cells from co-culture in presence of ECs is based on the biological capacity of ECs to adhere to plastic plates, and T cells that stay in suspension; hence, they were collected with gentle resuspension and aspiration.
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