The study protocol was approved by the institutional review board of Duksung Women's University (IRB No. 2017-002-001). ECFCs were isolated from the adherent mononuclear cell (MNC) fraction of human peripheral blood using CD31-coated magnetic beads (Invitrogen, MA, USA) as described previously [10 (link)]. The isolated ECFCs were expanded on 1% gelatin-coated plates (BD Biosciences, NJ, USA) using endothelial growth medium-2 (EGM-2 without hydrocortisone; Lonza, MD, USA) supplemented with 10% fetal bovine serum (FBS; Atlas Biologicals, CO, USA) and 1% glutamine-penicillin-streptomycin (GPS; Gibco, MA, USA). ECFCs between passages 7 and 10 were used for all experiments.
Cd31 coated magnetic beads
Manufactured by Thermo Fisher Scientific
Sourced in United States
CD31-coated magnetic beads are a type of lab equipment used for cell isolation and purification. These beads are coated with the CD31 (also known as PECAM-1) antibody, which binds to cells expressing the CD31 antigen, allowing for the selective isolation of these cells from a heterogeneous sample. The magnetic properties of the beads enable simple and efficient separation of the target cells using a magnetic field. These beads can be used in various cell biology and immunology applications that require the isolation of CD31-positive cells.
Automatically generated - may contain errors
Lab products found in correlation
Gelatin coated plates, by BD (4 mentions)
Fetal bovine serum (fbs), by Atlas Biologicals (3 mentions)
Glutamine penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Msc growth medium, by Lonza (2 mentions)
Egm mv 2 without hydrocortisone, by PromoCell (2 mentions)
Glutamine penicillin streptomycin gps, by Thermo Fisher Scientific (2 mentions)
Egm 2 without hydrocortisone, by Lonza (1 mentions)
Mscgm medium, by Lonza (1 mentions)
Fn coated plates, by Merck Group (1 mentions)
Ficoll paque, by GE Healthcare (1 mentions)
Egm 2, by Lonza (1 mentions)
Endothelial growth medium 2 egm 2, by Lonza (1 mentions)
6 protocols using cd31 coated magnetic beads
1
ECFC Isolation and Expansion Protocol
2
Isolation and Expansion of ECFCs and MSCs
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The study protocol was approved by the institutional review board of Duksung Women’s University (IRB No. 2017-002-01). The endothelial colony-forming cells (ECFCs) were isolated from human peripheral blood provided from one normal adult male donor. Some CD31-coated magnetic beads (Invitrogen, Waltham, MA, USA) were used to isolate ECFCs from the adherent mononuclear cell fraction of blood, as described in a previous report [19 (link)]. The isolated ECFCs were expanded on 1% gelatin-coated plates (BD Biosciences, Franklin Lakes, NJ, USA) using endothelial cell growth medium (EGM-2; Lonza, Basel, Switzerland) without hydrocortisone, supplemented with 10% fetal bovine serum (FBS; Atlas Biologicals, Fort Collins, CO, USA) and 1% glutamine–penicillin–streptomycin (GPS; Gibco, Waltham, MA, USA). Mesenchymal stem cells (MSCs) isolated from one normal human adult bone marrow were purchased from Lonza and then cultured using MSC growth medium (Lonza) supplemented with 10% FBS and 1% GPS. The ECFCs and MSCs between passages 7 and 10 were used for all experiments.
3
Isolation and Expansion of Human ECFCs
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Endothelial colony-forming cells (ECFCs) were isolated from the adherent mononuclear cell fraction of human peripheral blood using CD31-coated magnetic beads (Invitrogen, MA, USA) as previously described [11 (link)]. Isolated ECFCs were expanded on 1% gelatin-coated plates (BD Biosciences, NJ, USA) using an endothelial cell growth medium MV 2 (EGM-MV 2 without hydrocortisone; PromoCell, Heidelberg, Germany) supplemented with 10% fetal bovine serum (Atlas Biologicals, CO, USA) and 1% glutamine-penicillin-streptomycin (Gibco, MA, USA). ECFCs between passages seven and ten were used in all of the experiments. The protocol for this section of the study was approved by the institutional review board of Duksung Women's University (IRB Nos. 2017-002-001 and 2018-007-006).
4
Isolation and Expansion of ECFC and MPC
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Human umbilical cord blood was obtained from the Brigham and Women’s Hospital in accordance with an Institutional Review Board-approved protocol and according to the Declaration of Helsinki. Informed consent was not obtained as cord blood was excess human material, normally discarded, and was obtained without identifiers. ECFC were isolated from the adherent cell fraction using CD31-coated magnetic beads (Invitrogen) as described30 (link). ECFC were expanded on fibronectin (FN)-coated plates (1 μg/cm2; Millipore, MA) using EGM-2 (without hydrocortisone; Lonza) supplemented with 20% fetal bovine serum (FBS; Hyclone) and 1x glutamine-penicillin-streptomycin (GPS; Cellgro). ECFC between passages 5 and 8 were used for all experiments. ECFC express CD31, VE-cadherin, VEGFR-2, but not CD90, CD45 or CD143 (link), 31 (link).
MPC were isolated from the MNC fraction of human adult bone marrow (Lonza). MNC fraction isolated using Ficoll-Paque (GE Healthcare) was seeded on 1% gelatin-coated plates using MSCGM medium (Lonza) supplemented to 10% FBS, 1x GPS. Unbound cells were removed at 48 hours, and the adherent cell fraction maintained in culture until 70% confluent. MPC were expanded in MSCGM medium supplemented to 10% FBS, 1x GPS. MPC between passages 5 and 8 were used for all experiments. MPC express CD90 and CD105 but not CD31, VE-cadherin, VEGFR-2, CD45 or CD143 (link), 31 (link).
MPC were isolated from the MNC fraction of human adult bone marrow (Lonza). MNC fraction isolated using Ficoll-Paque (GE Healthcare) was seeded on 1% gelatin-coated plates using MSCGM medium (Lonza) supplemented to 10% FBS, 1x GPS. Unbound cells were removed at 48 hours, and the adherent cell fraction maintained in culture until 70% confluent. MPC were expanded in MSCGM medium supplemented to 10% FBS, 1x GPS. MPC between passages 5 and 8 were used for all experiments. MPC express CD90 and CD105 but not CD31, VE-cadherin, VEGFR-2, CD45 or CD143 (link), 31 (link).
5
Isolation and Expansion of Endothelial Colony-Forming Cells
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The study protocol was approved by the Institutional Review Board (IRB) of Duksung Women's University (IRB No. 2017-002-001, 2018-007-006). ECFCs were isolated from the adherent mononuclear cell fraction of human peripheral blood using CD31-coated magnetic beads (Invitrogen, Waltham, MA, USA) as described previously.14 (link) Isolated ECFCs were expanded on 1% gelatin-coated plates (BD Biosciences, Franklin Lakes, NJ, USA) using EC growth medium MV 2 (EGM-MV 2 without hydrocortisone; Promocell, Heidelberg, Germany) supplemented with 10% fetal bovine serum (FBS; Atlas Biologicals, Fort Collins, CO, USA) and 1% glutamine-penicillin-streptomycin (GPS; Gibco, Waltham, MA, USA). ECFCs obtained between passages 7 and 10 were used for all experiments.
6
Isolation and Expansion of ECFCs and MSCs
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The study protocol was approved by the institutional review board of Duksung Women’s University (IRB No. 2017-002-001). Human peripheral blood was provided from the national biobank. ECFCs were isolated from the adherent mononuclear cell (MNC) fraction using CD31-coated magnetic beads (Invitrogen, MA, USA) as described in the previous report (Melero-Martin et al., 2008 (link)). The isolated ECFCs were expanded on 1% gelatin-coated plates (BD Biosciences, NJ, USA) using endothelial growth medium-2 (EGM-2; Lonza, MD, USA) without hydrocortisone supplemented with 10% fetal bovine serum (FBS; Atlas Biologicals, CO, USA) and 1% glutamine-penicillin-streptomycin (GPS; Gibco, MA, USA). In all experiments, ECFCs from passages 7 to 10 were used.
MSCs were obtained from the MNC fraction of human adult bone marrow (Lonza). MSCs were cultured in MSC growth medium (Lonza) containing 10% FBS and 1% GPS until 80% confluence was achieved. MSCs from passage numbers 5 to 8 were used.
MSCs were obtained from the MNC fraction of human adult bone marrow (Lonza). MSCs were cultured in MSC growth medium (Lonza) containing 10% FBS and 1% GPS until 80% confluence was achieved. MSCs from passage numbers 5 to 8 were used.
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