Easysep human pan dc pre enrichment kit

Manufactured by STEMCELL

Sourced in Canada

The EasySep™ Human Pan-DC Pre-Enrichment Kit is a magnetic-based cell separation product designed to isolate dendritic cells from human cell samples. The kit utilizes a negative selection approach to enrich for the target cell population while removing unwanted cells.

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Lab products found in correlation

Facsaria, by BD (2 mentions) Ficoll paque plus, by GE Healthcare (2 mentions) Pan dc enrichment kit, by Miltenyi Biotec (1 mentions) Easysep monocyte enrichment kit, by STEMCELL (1 mentions) Ficoll paque, by GE Healthcare (1 mentions) Cd4 t cell isolation kit, by STEMCELL (1 mentions) Fitc anti human cd14, by Miltenyi Biotec (1 mentions) Fitc anti human cd16, by BD (1 mentions) Facsaria 3, by BD (1 mentions) Lymphoprep, by STEMCELL (1 mentions) Moflo astrios cell sorter, by Beckman Coulter (1 mentions) Lymphoprep, by Axis-Shield (1 mentions) Immunization grade bovine type 2 collagen, by Chondrex (1 mentions) Cpg odn 1826 tlrl 1826, by InvivoGen (1 mentions) Axl pe, by R&D Systems (1 mentions) Cd45ra vioblue, by Miltenyi Biotec (1 mentions) Cd1c percp efluor 710, by Thermo Fisher Scientific (1 mentions) Cd141 bv711, by BD (1 mentions) Diva software, by BD (1 mentions) Ficoll hypaque, by Thermo Fisher Scientific (1 mentions)

12 protocols using easysep human pan dc pre enrichment kit

Single-cell RNA-seq of human dendritic cells

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Cells were sorted on a FACSAria (BD biosciences). For SmartSeq2 scRNAseq, single cells were index sorted using the indicated gating strategy, from FT7 cultures differentiated on OP9+OP_DLL1 for 18 days. For the 10X Genomics scRNAseq experiments from cultures, cells were harvested by pipetting, filtered through a 30 μm nylon mesh, and sorted by flow cytometry as live (LIVE/DEAD Fixable Aqua Dead^-) total hematopoietic (CD45⁺) or Lin^- HLA-DR⁺, using PE-CD45, or FITC-lineage cocktail (CD3, CD14, CD16, CD19, CD20, CD56) with BV786-HLA-DR, APC-BDCA2/CD303 and V450-CD11c, before loading into the Chromium Single Cell Controller apparatus. For the 10X Genomics scRNAseq experiment with cells enriched from adult PBMCs, cells from a healthy blood donor were separated by density gradient centrifugation, enriched by magnetic bead-based sorting using the pan-DC enrichment kit from Miltenyi, stained and sorted for Lin^- HLA-DR⁺ cells as done for the cells from cultures, before loading into the Chromium Single Cell Controller apparatus. Peripheral blood XCR1^- and XCR1⁺ cDC1 subsets were sorted from PBMCs enriched with the EasySep Human Pan-DC Pre-Enrichment Kit (stem cell technologies), by gating on Lin^- HLA-DR⁺ CD123^- CD45RA^- CD11c⁺ CD1c^- SIRPa^- CD141⁺ CLEC9A⁺ CADM1⁺ cells. Sorted cells were cultured for 14 days on OP9_DLL1 by plating 10,000 to 30,000 cells/ml with the FT7+G cytokine cocktail.

Balan S., Arnold-Schrauf C., Abbas A., Couespel N., Savoret J., Imperatore F., Villani A.C., Vu Manh T.P., Bhardwaj N, & Dalod M. (2018). Large-Scale Human Dendritic Cell Differentiation Revealing Notch-Dependent Lineage Bifurcation and Heterogeneity. Cell Reports, 24(7), 1902-1915.e6.

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Isolation of Human Monocytes and DCs

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The isolation of human monocytes and dendritic cells from de-identified human subjects was performed under IRB Protocol #2015-2437 and through the UC Irvine ICTS Blood Donor Program. Donor sex: CD14 M = 2 males, 3 females. CD16 M = 2 males, 2 females. Blood DCs were all from female donors. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors using Ficoll-paque (GE Healthcare) gradient separation. In brief, blood was layered on top of Ficoll-paque and centrifuged in swinging bucket rotator without brake (400 × g, 40 minutes, 18°C). After centrifugation, plasma and upper layers were removed and PBMCs isolated from the interphase. Cells were then washed once with ice-cold PBS and used immediately. CD14 and CD16 monocytes were isolated via negative selection from PBMCs using the EasySep™ Monocyte Enrichment Kit (Stemcell Technologies) per manufacturer’s instructions. Blood dendritic cells were isolated via negative selection from PBMCs using the EasySep™ Human Pan-DC Pre-Enrichment Kit (Stemcell Technologies) per manufacturer’s instructions. Isolated cells were washed three times with PBS and sorted by FACS for either RNA-sequence analysis or used for further macrophage differentiation.

Abud E.M., Ramirez R.N., Martinez E.S., Healy L.M., Nguyen C.H., Newman S.A., Yeromin A.V., Scarfone V.M., Marsh S.E., Fimbres C., Caraway C.A., Fote G.M., Madany A.M., Agrawal A., Kayed R., Gylys K.H., Cahalan M.D., Cummings B.J., Antel J.P., Mortazavi A., Carson M.J., Poon W.W, & Blurton-Jones M. (2017). iPSC-derived human microglia-like cells to study neurological diseases. Neuron, 94(2), 278-293.e9.

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Isolation and Purification of Human DCs and CD4+ T Cells

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Peripheral blood mononuclear cells (PBMCs) were isolated by centrifugation on a density gradient (Lymphoprep, 07801, StemCell Technologies) following the manufacturer's protocol. Primary blood DCs were purified according to an established protocol (Alculumbre and Pattarani, 2016 (link)). In brief, total PBMCs were enriched in DCs using the EasySep Human Pan-DC Pre-Enrichment kit (StemCell Technologies). Enriched DCs were then sorted to obtain 98% purity on an MoFlo Astrios cell sorter (Beckman Coulter) or FACS ARIA III (BD Technologies), as lineage⁻ (CD3, CD14, CD16, CD56, CD20 and CD19; FITC anti-human CD3e, BD, 555339; FITC anti-human CD14, Miltenyi Biotec, 130-080-701; FITC anti-human CD16, BD, 347523; FITC anti-human CD56, BioLegend, 318304; FITC anti-human CD20, BD, 555622; FITC anti-human CD19, Miltenyi Biotec, 130-113-168), CD4⁺ (BV-650 anti-human CD4, BioLegend, 317436), CD11c⁺ (PeCy7 anti-human CD11c, Biolegend, 337216) and CD1c⁺ (PerCP-eFluor710 anti-human CD1c, also known as BDCA1, Thermo Fisher Scientific, 46-0015-42).
After enrichment from total PBMCs using the CD4⁺ T cell isolation kit (StemCell Technologies), naive CD4⁺ T cells were magnetically isolated. Purity was at least 95%.

Korniotis S., Saichi M., Trichot C., Hoffmann C., Amblard E., Viguier A., Grondin S., Noel F., Mattoo H, & Soumelis V. (2022). GM-CSF-activated human dendritic cells promote type 1 T follicular helper cell polarization in a CD40-dependent manner. Journal of Cell Science, 135(21), jcs260298.

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Isolation of Human CD1c+ Myeloid DCs

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Human CD1c⁺ myeloid DCs (mDCs) were isolated from blood samples obtained from patients with no known infections attending the Haemochromatosis Clinic in St. James’s Hospital after informed consent, as approved by the St. James’s Hospital and Tallaght University Hospital Joint Research Ethics Committee. First, PBMCs were separated by density gradient centrifugation on Lymphoprep™ (Axis-Shield). Cells were re-suspended in DC isolation buffer (2mM EDTA, 0.05% BSA in PBS) and DCs were enriched from PBMCs by negative magnetic separation using the EasySep™ Human Pan-DC Pre-Enrichment Kit following the manufacturer’s instructions (STEMCELL Technologies). The negative fraction containing DCs was then stained in DC isolation buffer with antibodies against lineage markers (Lin) (CD3, CD14, CD16, CD19, CD20, CD56), HLA-DR, CD1c and CD304 for 20min on ice in the dark. CD1c⁺ mDCs were further purified by cell sorting according to the following staining: Lin^-HLA-DR⁺CD304^-CD1c⁺.

Triglia D., Gogan K.M., Keane J, & O’Sullivan M.P. (2023). Glucose metabolism and its role in the maturation and migration of human CD1c+ dendritic cells following exposure to BCG. Frontiers in Cellular and Infection Microbiology, 13, 1113744.

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Purification and Immunization Protocol

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All flow antibodies were purchased from BD Biosciences and BioLegend of USA. And all ELISA kits were purchased from NEO Biosciences of China. EasySep™ Human Pan-DC Pre-Enrichment Kit for DC purification was purchased from STEMCELL of Canada and the purity of the DC up to 86.6 % in the research (Fig. S1). Immunization Grade Bovine Type II Collagen, immunization Grade Chick Type II Collage, Complete and incomplete Freund's adjuvants were purchased from Chondrex, USA. CpG ODN 1826 (tlrl-1826) and recombinant murine GM-CSF (AF-315-03) were purchased from InvivoGen and PeproTech of USA.

Han J., Li X., Luo X., He J., Huang X., Zhou Q., Han Y., Jie H., Zhuang J., Li Y., Yang F., Zhai Z., Wu S., He Y., Yang B, & Sun E. (2020). The mechanisms of hydroxychloroquine in rheumatoid arthritis treatment: Inhibition of dendritic cell functions via Toll like receptor 9 signaling. Biomedicine & Pharmacotherapy, 132, 110848.

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Profiling Myeloid Cells in HIV-1 Infection

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Peripheral blood mononuclear cells (PBMC) were isolated from HIV-1-infected patients (approved by Comité de protection des personnes CPP, ID-RCB 2017-A02820-53) or from controls (approved by the Institut National de la Santé et de la Recherche Médicale ethics committee) with Ficoll-Paque PLUS (GE). Informed consent was obtained from all donors, and samples were de-identified prior to use in the study. Total blood DCs were enriched with EasySep human pan-DC pre-enrichment kit (Stemcell Technologies 19251). DC enriched fraction were stained with antibodies specific for HLA-DR APCeFluor780, CD1c PerCPeFluor710 (eBioscience), CD123 Viogreen, CD45RA Vioblue, CD169 (Siglec-1) PE-Vio770 (Miltenyi), Axl PE (Clone #108724, R&D Systems), CD33 PE-CF594, CD141 BV711 (BD) and with a cocktail of antibodies against lineage markers CD19 (Miltenyi), CD3, CD14, CD16 and CD34 (BD) in the FITC channel. Data was acquired on a on a FACS Aria using Diva software (BD) and analysed in FlowJo v10 and GraphPad prism v7 for Mac.

Brouiller F., Nadalin F., Bonté P.E., Ait-Mohamed O., Delaugerre C., Lelièvre J.D., Ginhoux F., Ruffin N, & Benaroch P. (2023). Single-cell RNA-seq analysis reveals dual sensing of HIV-1 in blood Axl+ dendritic cells. iScience, 26(2), 106019.

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Isolation of Peripheral Blood Mononuclear Cells and Dendritic Cells

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Human blood (15-20 mL) collected in heparin tubes was diluted with PBS (human osmolarity) and underlayed with 10 mL Ficoll-Hypaque 1.077 g/mL density medium (Thermofisher Scientific, Waltham, Massachusetts) at RT. Samples were then centrifuged at 400 g for 30 min at 20°C with the brake off. The peripheral blood mononuclear cell (PBMC) layer was then collected, washed twice and counted. DCs were negatively selected from PBMCs using EasySep™ Human Pan-DC pre-enrichment kit (STEMCELL™ Technologies, Tullamarine, Victoria) according to manufacturer’s instructions.

Pang E.S., Daraj G., Balka K.R., De Nardo D., Macri C., Hochrein H., Masterman K.A., Tan P.S., Shoppee A., Magill Z., Jahan N., Bafit M., Zhan Y., Kile B.T., Lawlor K.E., Radford K.J., Wright M.D, & O’Keeffe M. (2022). Discordance in STING-Induced Activation and Cell Death Between Mouse and Human Dendritic Cell Populations. Frontiers in Immunology, 13, 794776.

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Enrichment of CD135+ and Human Myeloid Cells

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Mouse cell suspensions were incubated with anti-CD135-biotin (Biolegend)
for 30 min at 4°C, washed and incubated with Ultra-Pure anti-Biotin
microbeads (Miltenyi) for 30 min at 4°C. CD135⁺ cells were
selected using LS columns (Miltenyi). Human myeloid cells were negatively
enriched from PBMCs using anti-CD3 (OKT3), anti-CD14 (HCD14), anti-CD19 (HIB19)
and anti-CD335 (9E2) Abs followed by anti-mouse Dynabeads (Invitrogen) at a
concentration of 4 beads per target cell⁵². For IL-1β experiments, human DC were negatively
enriched using EasySep Human pan-DC Pre Enrichment Kit (StemCell
Technologies).

Sulczewski F.B., Maqueda-Alfaro R.A., Alcántara-Hernández M., Perez O.A., Saravanan S., Yun T.J., Seong D., Hornero R.A., Raquer-McKay H.M., Esteva E., Lanzar Z.R., Leylek R.A., Adams N.M., Das A., Rahman A.H., Gottfried-Blackmore A., Reizis B, & Idoyaga J. (2023). TRANSITIONAL DENDRITIC CELLS ARE DISTINCT FROM CONVENTIONAL DC2 PRECURSORS AND MEDIATE PRO-INFLAMMATORY ANTIVIRAL RESPONSES. Nature immunology, 24(8), 1265-1280.

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Dendritic Cell Isolation and Coculture

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The PBMCs prepared in Section 2.1 were resuspended in RoboSep Buffer (STEMCELL, USA) and used immediately. DCs were isolated by negative immunoselection with an EasySep Human Pan‐DC Pre‐Enrichment Kit (STEMCELL, Canada) according to the manufacturer's instructions. To increase the DC ratio, the magnetic particle binding step was performed twice. After isolation, the cells were resuspended in RPMI‐1640 supplemented with 25 mM HEPES, 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. The cells (1 × 10⁵ cells/well) were incubated in a 384‐well plate (Corning, USA) and cocultured with 1 × 10⁶ cells/well of hk‐MCC1849 or hk‐ATCC53103 at 37°C under 5% CO2 and humid conditions. A no‐addition well was used as the control.

Li Y., Aoki T., Iwabuchi S., Arai S., Iwabuchi N., Motobayashi H., Tanaka M, & Hashimoto S. (2024). Immunomodulatory activity of heat‐killed Lacticaseibacillus paracasei MCC1849 based on the activation of plasmacytoid dendritic cells in the peripheral blood of healthy adults. Food Science & Nutrition, 12(5), 3452-3460.

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Isolation of Human Blood Dendritic Cells

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Blood dendritic cells were isolated from human whole blood as described previously (Jain et al. 2007 (link)). Briefly, buffy coat isolated from whole blood from normal donors was purchased from Biological Specialty Corporation (Colmar, PA). Peripheral blood mononuclear cells (PBMCs) were then isolated from the buffy coat via density centrifugation using Ficoll-Paque Plus (GE, Pittsburg, PA). Isolated PBMCs were washed with hank’s balanced salt solution (HyClone, GE) followed by treatment with red blood cell (RBC) lysis buffer (Biolegend, San Diego, CA) for removal of any RBCs that were carried over from the buffy coat. DCs were subsequently enriched by negative selection using EasySep™ human pan-DC pre-enrichment kit (Stemcell Technologies, Vancouver Canada) as per the manufacturer’s protocol.

Ginwala R., Bhavsar R., Moore P., Bernui M., Singh N., Bearoff F., Nagarkatti M., Khan Z.K, & Jain P. (2020). Apigenin modulates dendritic cell activities and curbs inflammation via RelB inhibition in the context of neuroinflammatory diseases. Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology, 16(2), 403-424.

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