The isolation procedure of endometrial cells was carried out as described (5) . The tissues were minced and digested with PBS containing collagenase type III (0.3 mg/ml, Worthington Biochemical Corporation, NJ, USA) and deoxyribonuclease type I (40 μg/ml, Worthington Biochemical Corporation) for one hour at 37 o C. Red blood cells were removed using Ficoll-Paque (GE Healthcare, Uppsala, Sweden) densitygradient centrifugation. Leukocytes were excluded using anti-CD45 antibody-coated Dynabeads (Invitrogen, Waltham, MA, USA). Epithelial cells were separated from the stromal cells using anti-CD326 (EpCAM) antibody-coated microbeads (Miltenyi Biotec Inc., San Diego, CA, USA). Freshly isolated epithelial cells and stromal cells were used for coculture and collection of condition medium as describe below. Some of the spare puri ed stromal cells (6000-8000 cells/cm 2 ) were plated into 100-mm petri dishes coated with bronectin (1 mg/ml, Invitrogen) and cultured in growth medium containing 10% FBS (Invitrogen), 1% antibiotics (Invitrogen) and 2 mmol/L glutamine (Invitrogen) in DMEM/F12 (Sigma-Aldrich, St Louis, MA, USA) for 7-14 days in a humidi ed carbon dioxide incubator at 37 o C in 5% CO 2 . The medium was changed every seven days until the cells reached 90% con uence.
Anti cd326 epcam antibody coated microbeads
Manufactured by Miltenyi Biotec
Sourced in United States
Anti-CD326 (EpCAM) antibody-coated microbeads are laboratory equipment used for the isolation and enrichment of epithelial cells expressing the CD326 (EpCAM) antigen. These microbeads are designed to facilitate the separation and purification of target cells from complex samples such as blood, tissue, or cell suspensions.
Automatically generated - may contain errors
Lab products found in correlation
Deoxyribonuclease type 1, by Worthington (5 mentions)
Collagenase type 3, by Worthington (5 mentions)
Ficoll paque, by GE Healthcare (5 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Dmem f12, by Merck Group (5 mentions)
Anti cd45 antibody coated dynabeads, by Thermo Fisher Scientific (3 mentions)
Fibronectin, by Thermo Fisher Scientific (3 mentions)
Bronectin, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Dynabead, by Thermo Fisher Scientific (2 mentions)
Glutamine, by Thermo Fisher Scientific (1 mentions)
5 protocols using anti cd326 epcam antibody coated microbeads
1
Endometrial Cell Isolation and Culture
2
Isolation of Endometrial Stromal Cells
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The isolation of single endometrial stromal cells was performed according to our previous study17 (link). In brief, endometrial tissue was minced into small pieces and digested with PBS containing collagenase type III (0.3 mg/ml, Worthington Biochemical Corporation, NJ, USA) and deoxyribonuclease type I (40 μg/ml, Worthington Biochemical Corporation) at 37 °C for 1 h. After two rounds of digestion, Ficoll-Paque (GE Healthcare, Uppsala, Sweden) centrifugation and anti-CD45 antibody coated Dynabeads (Invitrogen, Waltham, MA, USA) were sequentially used to remove the red blood cells and the leukocytes, respectively. The stromal cells were then separated from the epithelial cells using anti-CD326 (EpCAM) antibody-coated microbeads (Miltenyi Biotec Inc., San Diego, CA, USA). Next, freshly isolated stromal cells were seeded into 100 mm dishes coated with fibronectin (1 mg/ml, Gibco) and cultured in growth medium (GM) containing 10% FBS (Invitrogen), 1% L-glutamine (Invitrogen), and 1% penicillin-streptomycin (Invitrogen) in DMEM/F-12 (Sigma-Aldrich, St Louis, MA, USA). Stromal cells were cultured in a humidified carbon dioxide incubator at 37 °C. The medium was changed every 7 days until the cells reached 80% confluence.
3
Isolation of Endometrial Epithelial and Stromal Cells
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The isolation procedure of endometrial cells was carried out as described [5 (link)]. The tissues were minced and digested with PBS containing collagenase type III (0.3 mg/ml, Worthington Biochemical Corporation, NJ, USA) and deoxyribonuclease type I (40 μg/ml, Worthington Biochemical Corporation) for 1 h at 37 °C. Red blood cells were removed using Ficoll-Paque (GE Healthcare, Uppsala, Sweden) density-gradient centrifugation. Leukocytes were excluded using anti-CD45 antibody-coated Dynabeads (Invitrogen, Waltham, MA, USA). Epithelial cells were separated from the stromal cells using anti-CD326 (EpCAM) antibody-coated microbeads (Miltenyi Biotec Inc., San Diego, CA, USA). Freshly isolated epithelial cells and stromal cells were used for coculture and collection of condition medium as describe below. Some of the spare purified stromal cells (6000–8000 cells/cm2) were plated into 100-mm petri dishes coated with fibronectin (1 mg/ml, Invitrogen) and cultured in growth medium containing 10% FBS (Invitrogen), 1% antibiotics (Invitrogen) and 2 mmol/L glutamine (Invitrogen) in DMEM/F12 (Sigma-Aldrich, St Louis, MA, USA) for 7–14 days in a humidified carbon dioxide incubator at 37 °C in 5% CO2. The medium was changed every 7 days until the cells reached 90% confluence.
4
Isolation of Endometrial Stromal Cells
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+ Expand
The isolation of single endometrial stromal cells was performed according to our previous study [28 (link)]. In brief, endometrial tissue was minced into small pieces and digested with PBS containing collagenase type III (0.3 mg/mL, Worthington Biochemical Corporation, NJ, USA) and deoxyribonuclease type I (40 μg/mL, Worthington Biochemical Corporation, NJ, USA) at 37 °C for 1 h. After two rounds of digestion, Ficoll-Paque (GE Healthcare, Uppsala, Sweden) centrifugation and anti-CD45 antibody coated Dynabeads (Invitrogen, Waltham, MA, USA) were sequentially used to remove the red blood cells and the leukocytes, respectively. Purified stromal cells were then separated from epithelial cells using anti-CD326 (EpCAM) antibody-coated microbeads (Miltenyi Biotech, San Diego, CA, USA). Next, freshly isolated stromal cells were seeded into 100 mm dishes coated with fibronectin (1 mg/mL, Life Technologies, Carlsbad, CA, USA) and cultured in growth medium (GM) containing 10% FBS (Invitrogen, Waltham, MA, USA), 1% L-glutamine (Invitrogen, Waltham, MA, USA) and 1% penicillin-streptomycin (Invitrogen, Waltham, MA, USA) in DMEM/F-12 (Sigma-Aldrich, St Louis, MA, USA). Stromal cells were cultured in a humidified carbon dioxide incubator at 37 °C. The medium was changed every 7 days until the cells reached 80% confluence.
5
Isolation of Endometrial Cell Subsets
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The isolation procedure of endometrial cells was carried out as described [5] . The tissues were minced and digested with PBS containing collagenase type III (0.3 mg/ml, Worthington Biochemical Corporation, NJ, USA) and deoxyribonuclease type I (40 μg/ml, Worthington Biochemical Corporation) for one hour at 37 o C. Red blood cells were removed using Ficoll-Paque (GE Healthcare, Uppsala, Sweden) densitygradient centrifugation. Leukocytes were excluded using anti-CD45 antibody-coated Dynabeads (Invitrogen, Waltham, MA, USA). Epithelial cells were separated from the stromal cells using anti-CD326 (EpCAM) antibody-coated microbeads (Miltenyi Biotec Inc., San Diego, CA, USA). Freshly isolated epithelial cells and stromal cells were used for coculture and collection of condition medium as describe below. Some of the spare puri ed stromal cells (6000-8000 cells/cm 2 ) were plated into 100-mm petri dishes coated with bronectin (1 mg/ml, Invitrogen) and cultured in growth medium containing 10% FBS (Invitrogen), 1% antibiotics (Invitrogen) and 2 mmol/L glutamine (Invitrogen) in DMEM/F12 (Sigma-Aldrich, St Louis, MA, USA) for 7-14 days in a humidi ed carbon dioxide incubator at 37 o C in 5% CO 2 . The medium was changed every seven days until the cells reached 90% con uence.
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