Cd326 epcam microbeads

Manufactured by Miltenyi Biotec

Sourced in Germany

CD326 (EpCAM) MicroBeads are magnetic beads coated with antibodies that specifically recognize the CD326 (EpCAM) antigen. These beads can be used for the isolation and enrichment of cells expressing the CD326 antigen from various sample types.

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Lab products found in correlation

Ls column, by Miltenyi Biotec (3 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions) Low gelling temperature agarose, by Merck Group (1 mentions) Dispase, by Corning (1 mentions) Cd31 specific magnetic beads, by Miltenyi Biotec (1 mentions) Low melt agarose, by Merck Group (1 mentions) Dispase, by BD (1 mentions) Dnase 1, by AppliChem (1 mentions) Cd144, by Miltenyi Biotec (1 mentions) Anti integrin α 7 microbeads, by Miltenyi Biotec (1 mentions) Ld column, by Miltenyi Biotec (1 mentions) Microbead, by Miltenyi Biotec (1 mentions) Cd45 microbeads, by Miltenyi Biotec (1 mentions) Sb202190, by Bio-Techne (1 mentions) A83 01, by Bio-Techne (1 mentions) Rhfgf, by R&D Systems (1 mentions) Rhvegf, by R&D Systems (1 mentions) Rhbmp 4, by R&D Systems (1 mentions) Accutase, by Innovative Cell Technologies (1 mentions) Rock inhibitor y 27632 dihydrochloride, by Bio-Techne (1 mentions)

15 protocols using cd326 epcam microbeads

Isolation of Alveolar Type II Cells

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The pmATII cells were isolated as previously described [28 (link),32 (link)] with slight modifications. In brief, lungs were filled with dispase (Corning, New York, NY, USA) and low gelling temperature agarose (Sigma Aldrich, Saint Louis, MO, USA) before tissue was minced and the cell suspension was filtered through 100-, 20-, and 10-μm nylon meshes (Sefar, Heiden, Switzerland). Negative selection of fibroblasts was performed by adherence on non-coated plastic plates. Macrophages and white blood cells were depleted with CD45 and endothelial cells were depleted with CD31 specific magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instructions. Cell purity was assessed routinely by analysis of endothelial (CD31), mesenchymal (α-SMA, CD90), epithelial (EpCAM, panCK and proSP-C), and hematopoietic cell (CD45) markers by immunofluorescence or flow cytometry.
For the analysis of WNT-GFP epithelial cells and for the organoid experiments, isolation was performed as described above. No depletion of fibroblasts was performed, the CD45 and CD31 depleted single cell suspension was further enriched for epithelial cells by positive selection using EpCAM (CD326) Microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany).

Lehmann M., Hu Q., Hu Y., Hafner K., Costa R., van den Berg A, & Königshoff M. (2020). Chronic WNT/β-catenin signaling induces cellular senescence in lung epithelial cells. Cellular signalling, 70, 109588.

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Isolation of Lung Epithelial (EpCAM+) Cells

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Epithelial (EpCAM⁺) cells were isolated from lungs of 8–12 week old male and female C57Bl6 mice with microbeads as described previously (Ng-Blichfeldt et al., 2018 (link); Ng-Blichfeldt et al., 2019 (link); Wu et al., 2019a (link)). Lungs of mice were flushed through the heart with PBS, instilled with dispase (BD Biosciences, Oxford, United Kingdom, #354235) and low-melt agarose (Sigma Aldrich, Poole, United Kingdom #A9414), and incubated at room temperature (RT) for 45 min. Trachea and extrapulmonary airways were removed, and the remaining lobes were homogenized in DMEM medium with dnase1 (Applichem, Germany #A3778). The resulting suspension was passed through a cell strainer with the size of 100 μm, incubated with microbeads conjugated to antibodies for CD45 (Miltenyi Biotec, Teterow, Germany #130–052–301) and CD31 (Miltenyi, #130–097–418), and passed through LS columns (Miltenyi #130–091–051). The CD31^-/CD45^- suspension was then enriched for epithelial cells by positive selection using EpCAM (CD326) microbeads (Miltenyi #130–105–958). EpCAM⁺ cells were resuspended in DMEM with 10% FBS.

Wu X., Verschut V., Woest M.E., Ng-Blichfeldt J.P., Matias A., Villetti G., Accetta A., Facchinetti F., Gosens R, & Kistemaker L.E. (2021). Rho-Kinase 1/2 Inhibition Prevents Transforming Growth Factor-β-Induced Effects on Pulmonary Remodeling and Repair. Frontiers in Pharmacology, 11, 609509.

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Isolation of Lizard Dermal Fibroblasts

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Lizard (L. lugubris) tail and limb tissues were washed three times in 10% povidone-iodine solution and washed once in HBSS supplemented with 100 units/mL penicillin, 100 µg/mL streptomycin, and 250 ng/mL fungizone antimycotic. Washed tissues were incubated in 0.1% EDTA in HBSS for 45 min at room temperature with agitation, and scales/epidermis were peeled from each tissue piece with forceps and discarded. Prepared tissues were then washed extensively in HBSS, minced, and digested in 1 mg/mL trypsin and 1 mg/mL collagenase II for 1 h at 37 °C. Immune, muscle-related, and endothelial cells were depleted by passing cell suspensions through the following MACS® (Miltenyi Biotec) magnetic beads: CD144 (VE-Cadherin) MicroBeads (PN: 130-097-857), Anti-Integrin α−7 MicroBeads (PN: 130-104-26), CD45 MicroBeads (PN: 130-052-301), CD326 (EpCAM) MicroBeads (PN: 130-105-958), according to the manufacturer’s instructions. Cell/bead suspensions were loaded onto LD columns (Miltenyi Biotec, PN: 130-042-901) and placed on MidiMACS™ Separator magnets (Miltenyi Biotec, PN: 130-042-301). Enriched fibroblast suspensions were collected in lizard cell culture medium (Dulbecco’s Modified Eagle Media (DMEM)/Ham’s F12, 2 mM Glutamax, 0.1 µM dexamethasone, 40 µg/mL proline, 50 µg/mL ascorbate, and 10 µg/mL ITS+ supplement).

Vonk A.C., Zhao X., Pan Z., Hudnall M.L., Oakes C.G., Lopez G.A., Hasel-Kolossa S.C., Kuncz A.W., Sengelmann S.B., Gamble D.J, & Lozito T.P. (2023). Single-cell analysis of lizard blastema fibroblasts reveals phagocyte-dependent activation of Hedgehog-responsive chondrogenesis. Nature Communications, 14, 4489.

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Isolation and Culture of Intestinal Epithelial Organoids

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HIOs were dissociated to a single cell suspension as described previously. Dissociated cells were incubated with CD326/EpCAM MicroBeads (Miltenyl Biotec, San Diego, CA, USA) for 30 minutes at 4 °C, and EpCAM+ and EpCAM– populations of cells were obtained via MACS. EpCAM+ cells were cultured as epithelial-only HIOs (eHIOs) as previously described.¹³ Briefly, EpCAM+ cells were maintained in organoid media supplemented with SB202190 (10 µM; Tocris) and A83-01 (500nM; Tocris). EPCAM– cells were cultured in advanced Dulbecco’s modified Eagle medium media containing 10% fetal calf serum and were passaged weekly.

Estrada H.Q., Patel S., Rabizadeh S., Casero D., Targan S.R, & Barrett R.J. (2021). Development of a Personalized Intestinal Fibrosis Model Using Human Intestinal Organoids Derived From Induced Pluripotent Stem Cells. Inflammatory Bowel Diseases, 28(5), 667-679.

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Mesoderm Induction and hEMP Isolation

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Mesoderm commitment was induced as previously described^{9 (link),79 (link),80 (link)} with certain optimizations. Briefly, hESC cells were maintained on Matrigel-coated 6-well plates in mTeSR Plus complete medium. At day (D) −18, mesoderm induction was initiated in X-VIVO 15 medium (Lonza, Cat. 04–418Q) supplemented with recombinant human (rh) Activin A (10 ng/ml) (R&D Systems, Cat. 338-AC-010), rhBMP4 (10 ng/ml) (R&D Systems, Cat. 314-BP-010), rhVEGF (10 ng/ml) (R&D Systems, Cat. 298-VS-005), rhFGF (10 ng/ml) (R&D Systems, Cat. 233-FB-025), and ROCK inhibitor Y-27632 dihydrochloride (10 μM) (Tocris, Cat. 1254). hESCs were plated on Matrigel coated 6-well plates at 3×10⁶ cells per well in 3ml. Medium was then changed daily with X-VIVO 15 supplemented with rhBMP4 (10 ng/ml), rhVEGF (10 ng/ml), and rhFGF (10 ng/ml). At D-14, cells were washed with PBS and detached with Accutase (Innovative Cell Technologies, Cat. AT-104) (1 mL per well, for 10 min at 37°C). Cells were harvested and hEMPs isolated by depletion of CD326+ cells by magnetic cell sorting (MACS) using CD326 (EpCAM) MicroBeads (Miltenyi, Cat. 130–061-101).

Weinreich M., Liang C, & Stillman B. (1999). The Cdc6p nucleotide-binding motif is required for loading Mcm proteins onto chromatin. Proceedings of the National Academy of Sciences of the United States of America, 96(2), 441-446.

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Efficient Enrichment and Isolation of BECs

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In some experiments, we enriched BECs using a magnetic activated cell sorting (MACS) system (Miltenyi) for more efficient FACS sorting, while in other experiments we directly proceeded to staining for FACS. We confirmed that high purity of FACS-sorted BECs was achieved in each setting (YFP− EPCAM+ rate >90%). For MACS enrichment, the cells were resuspended in MACS buffer (PBS, 0.5% BSA, 2 mM EDTA) to the final volume of approximately 1.5 mL, and 150 μL CD326 (EPCAM) MicroBeads (Miltenyi) were added. After incubation at 4°C for 15 min, the cells were washed with an equal volume of MACS buffer, and then centrifuged at 2,000 rpm (∼800 × g) at 4°C for 2 min. The cells were resuspended in 1 mL MACS buffer, and EPCAM+ cells were separated using LS columns (Miltenyi) (2 columns were used per animal; 0.5 mL suspension/column). The cells were then collected by centrifugation at 2,000 rpm (∼800 × g) at 4°C for 2 min. The cells were then resuspended in 100 μL flow buffer(+), and approximately 1 μL BV421-EPCAM (Biolegend) on ice for 20 min. After washing in 200 μL flow buffer(+) once by centrifugation at 2,000 rpm (∼800 × g) at 4°C for 2 min, the cells were resuspended in 0.5 mL flow buffer(+) with 1/1,000 TO-PRO-3, and the cells were sorted on an Aria II sorter (BD).

Katsuda T., Sussman J., Li J., Merrell A.J., Vostrejs W., Secreto A., Matsuzaki J., Ochiya T, & Stanger B.Z. (2023). Evidence for in vitro extensive proliferation of adult hepatocytes and biliary epithelial cells. Stem Cell Reports, 18(7), 1436-1450.

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EpCAM-Based Cell Isolation and Sorting

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Cells were trypsinized, washed, and resuspended in Hank’s Balanced Salt Solution supplemented with 1% HEPES and 2% FBS. Cells were then incubated with an APC-conjugated anti-CD326 (EpCAM) on ice for 30 min, and EpCAM positive and negative cells were isolated using a BD FACSAria II cell sorting system (BD Biosciences). In addition, EpCAM⁺ and EpCAM⁻ cells were also sorted for functional studies using an autoMACS pro cell separator and CD326 (EpCAM) microbeads (Miltenyi Biotec K.K.).

Hayashi T., Yamashita T., Okada H., Nio K., Hara Y., Nomura Y., Hayashi T., Asahina Y., Yoshida M., Oishi N., Sunagozaka H., Takatori H., Honda M, & Kaneko S. (2017). Sporadic PCDH18 somatic mutations in EpCAM-positive hepatocellular carcinoma. Cancer Cell International, 17, 94.

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Isolation and Culture of Primary Renal Tubular Epithelial Cells

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Primary C57BL/6 renal tubular epithelial cells were isolated as described previously^{28 (link)}. Kidneys were digested using multi-tissue dissociation kit and GentleMacs (Miltenyi, Bergisch Gladbach, Germany), incubated with CD326 (EpCAM) microbeads (Miltenyi) and passed through LS columns. The positive cell fraction was suspended in defined K1 medium: DMEM/F12 medium supplemented with 25 ng/ml epidermal growth factor (Sigma Aldrich, St Louis, MO), 1 ng/ml prostaglandin E₁ (Cayman Chemicals, Ann Arbor, MI), 5 × 10⁻¹¹ M triiodothyronine (Sigma Aldrich), 5 × 10⁻⁸ M hydrocortisone (Sigma-Aldrich), insulin–transferrin–sodium selenite supplement (Sigma Aldrich), 1% penicillin/streptomycin (Thermofisher Scientific, Waltham, MA), 25 mM HEPES (Thermofisher Scientific), and 5% FCS (Thermofisher Scientific) and cultured on collagen-coated dishes (BD Biosciences, Franklin Lakes, NJ).
Cell passages 2–4 were used for experimental work. Primary RTEC cultures were treated for 24 h in the following groups: untreated, LPS only (1 μg/mL, InvivoGen; San Diego, USA), LPS + colchicine (1 μM), LPS + metformin (1 mM). In further experiments, RTEC were subjected to normoxia (FiO₂ 21%) or hypoxia (FiO₂ 1%) for 24 h. All drug treatments were administered 2 h prior to LPS stimulation or hypoxia. Cell lysates were collected for RNA and protein.

El-Rashid M., Nguyen-Ngo D., Minhas N., Meijles D.N., Li J., Ghimire K., Julovi S, & Rogers N.M. (2020). Repurposing of metformin and colchicine reveals differential modulation of acute and chronic kidney injury. Scientific Reports, 10, 21968.

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Lung Cell Isolation and Enrichment

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The right middle lobe of the lung was minced into small pieces and resuspended in DMEM F12 media (5 ml) containing 2.6 IU Liberase (Sigma-Aldrich) and 30 μg DNase I (Sigma-Aldrich). Tissue digestion was carried out under constant shaking at 37°C for 1 h. After incubation, the single cell suspension was made by pipetting up and down followed by filtering the cells through 70 micron and 40 micron nylon mesh to remove clumped cells and undigested tissues. Depletion of lung epithelial cells in single cells suspension was carried out using CD326 (EpCAM) MicroBeads (Miltenyi Biotec) and magnetically sorted through the LS column (Miltenyi Biotec). The single cell suspension devoid of lung epithelial cells was washed and counted using the Countess 3 Automated cell counter. The dissociated lung cells were resuspended in 100 μl of BD stain buffer (BD Bioscience) containing 1.0 × 10⁶ cells and blocked with anti-CD16/32 antibody before staining to block non-specific binding of staining antibody (BioLegend, San Diego CA).

Srinivasan A., Giri A., Duraisamy S.K., Alsup A., Castro M, & Sundar I.K. (2023). Chronic HDM exposure shows time-of-day and sex-based differences in inflammatory response associated with lung circadian clock disruption. iScience, 26(9), 107580.

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Breast Cancer Cell Culture Protocol

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Murine 4T1 cells and EMT6 cells were obtained from American Type Culture Collection (ATCC, USA) and were cultured in RPMI-1640 medium (Hyclone, USA) supplemented with 10% fetal bovine serum (ScienCell, USA) and 1% penicillin/streptomycin.
CAFs were isolated from cancer-associated regions of whole breast tissues. The dissociated tissues were minced with scissors and digested with collagenase type I (1 mg/ml; Sigma) and hyaluronidase (100 U/ml; Sigma) at 37 °C for 12 h in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 2% bovine serum albumin (BSA). The stromal cell-enriched supernatant was collected for centrifugation at 250 g for 10 min. The cell pellet was resuspended in DMEM/F12 medium supplemented with 10% fetal bovine serum, 10 ng/ml EGF (Peprotech, USA), and 10 ng/ml FGF (Peprotech, USA). We plated the cell suspension in cell culture flasks, which were left undisturbed for 2 to 5 days.
Primary BC cells were sorted using CD326 (EpCAM) MicroBeads (Miltenyi Biotech, Germany), according to the manufacturer’s instructions, for the positive selection of viable epithelial tumor cells from single-cell preparations from tissues of patients with breast carcinoma. The positively selected cells were suspended in DMEM/F12 medium supplemented with 10% fetal bovine serum.
All cell lines were cultured in a humidified incubator at 37 °C and 5% CO₂.

Ouyang L., Chang W., Fang B., Qin J., Qu X, & Cheng F. (2016). Estrogen-induced SDF-1α production promotes the progression of ER-negative breast cancer via the accumulation of MDSCs in the tumor microenvironment. Scientific Reports, 6, 39541.

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