Human pan dc pre enrichment kit

Manufactured by STEMCELL

Sourced in Canada

The Human Pan-DC Pre-Enrichment Kit is a laboratory product designed to enrich for dendritic cells from human peripheral blood mononuclear cells. The kit utilizes positive selection to isolate this cell population.

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Lab products found in correlation

Ficoll paque plus, by STEMCELL (2 mentions) Cd1c bdca1 percp cy5, by BioLegend (2 mentions) Hla dr apc eflour780 ln3, by Thermo Fisher Scientific (2 mentions) Cd11c v450 b ly6, by BD (2 mentions) Lineage cocktail 1 fitc, by BD (2 mentions) Diva software, by BD (2 mentions) Facsaria 2, by BD (2 mentions) Anti cd86 fitc, by BD (1 mentions) Alexafluor700 anti hla dr, by BioLegend (1 mentions) Glutamax, by Thermo Fisher Scientific (1 mentions) Lsrii instrument, by BD (1 mentions) Facsvantage, by BD (1 mentions) Facsaria 3, by BD (1 mentions) Rhtslp, by R&D Systems (1 mentions) Facsaria, by BD (1 mentions) Lps eb ultrapure, by Merck Group (1 mentions) Gmp dc medium, by CellGenix (1 mentions) Anti cd3, by Thermo Fisher Scientific (1 mentions) Ficoll paque plus, by GE Healthcare (1 mentions)

Close spelling variants of this product

EasySep™ Human Pan-DC Pre-Enrichment Kit Pan-DC Enrichment Kit

8 protocols using human pan dc pre enrichment kit

pDC Enrichment and Activation Assay

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In order to enrich plasmacytoid dendritic cells (pDCs), cells expressing CD3, CD9, CD14, CD16, CD19, CD34, CD56, CD66b, and glycophorin A were depleted from PBMCs using magnetic sorting (Human Pan-DC Pre-Enrichment Kit, StemCell Technologies). pDCs were then sorted on a FACS Vantage instrument (BD Biosciences). A representative gating strategy is provided in Supplementary Fig. 6b. pDCs were cultured for 24 h at 37 °C and 5% CO₂ with medium RPMI 1640 Medium, GlutaMAX (Life Technologies) with 10% FCS, GM-CSF (10 ng/mL) used as a positive control or DC supernatants. Cells were stained for 15 min at 4 °C using a FITC-anti-CD86 (BD, 1:20), an APC-anti-ICOSL (R&D Systems, 1:20) and Alexa-Fluor-700-anti-HLA-DR (Biolegend, 1:20) or with the corresponding isotypes. Cells were analyzed on an LSR II instrument (BD Biosciences).

Noël F., Massenet-Regad L., Carmi-Levy I., Cappuccio A., Grandclaudon M., Trichot C., Kieffer Y., Mechta-Grigoriou F, & Soumelis V. (2021). Dissection of intercellular communication using the transcriptome-based framework ICELLNET. Nature Communications, 12, 1089.

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Purification of Plasmacytoid and Myeloid Dendritic Cells

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Platelet apheresis residues from healthy donors were provided by the Etablissement Français du Sang of Nantes (agreement NTS-2014–06). DCs were obtained from peripheral blood mononuclear cells (PBMCs), as previously described.⁶⁰ (link) Briefly, DCs were enriched by counterflow centrifugation elutriation and then purified, using a human pan-DC pre-enrichment kit (StemCell Technologies, Inc.) as recommended by the manufacturer, which consists in an immunomagnetic negative isolation of DCs. Among the cells obtained, pDCs were stained with an APC-conjugated anti-BDCA-4 mAb (clone AD5–17F6, Miltenyi) and CD1c⁺ DCs with a FITC-conjugated anti-CD1c mAb (clone L161, eBiosciences) to sort them by flow cytometry (FACS Aria III, BD Biosciences). To improve the cell sorting, BV421-conjugated anti-CD3 (clone UCHT1), anti-CD19 (clone HIB19) and anti-CD14 (clone MφP9) mAbs (BD Biosciences) were used to eliminate other immune populations.

Achard C., Guillerme J.B., Bruni D., Boisgerault N., Combredet C., Tangy F., Jouvenet N., Grégoire M, & Fonteneau J.F. (2016). Oncolytic measles virus induces tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated cytotoxicity by human myeloid and plasmacytoid dendritic cells. Oncoimmunology, 6(1), e1261240.

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Isolation and Culture of Human Dendritic Cells

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DC were enriched from PBMC obtained after Ficoll-Paque Plus density gradient centrifugation (Stemcell Technologies; Vancouver, BC, Canada) by negative selection with monoclonal antibodies (mAb) to CD3, CD9, CD14, CD16, CD19, CD34, CD56, CD66b, and glycophorin A (Human pan-DC pre-enrichment kit; Stemcell Technologies). Cells were labeled with anti-human lineage cocktail-FITC (CD3, CD14, CD16, CD19, CD20 and CD56); CD123-PE (9F5) and CD11c-APC (S-HCL-3) (BD Biosciences; Franklin Lakes, NJ), and HLA-DR-APC-eflour780 (LN3) (Sigma-Aldrich; St. Louis, MO); lin⁻CD123⁻HLA-DR⁺CD11c⁺ DC were sorted with FACS Aria (BD Bioscience). DC were seeded at 100 × 10³ cells/well in 200 μl of RPMI with 10% human AB serum, and cultured with medium alone or in the presence of 20 ng/ml of rhTSLP (R&D), or tumor-derived products. After 48 hours DC were harvested, washed, and analyzed or used in experiments.

Wu T.C., Xu K., Banchereau R., Marches F., Yu C.I., Martinek J., Anguiano E., Pedroza-Gonzalez A., Snipes G.J., O’Shaughnessy J., Nishimura S., Liu Y.J., Pascual V., Banchereau J., Oh S, & Palucka K. (2014). Reprogramming tumor-infiltrating dendritic cells for CD103+CD8+ mucosal T cell differentiation and breast cancer rejection. Cancer immunology research, 2(5), 487-500.

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Isolation of PBMCs and DCs

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PBMCs were isolated from leukapheresis products using Ficoll-Paque Plus density gradient centrifugation (Stemcell Technologies, Vancouver, BC, Canada). DCs were first enriched using human Pan-DC Pre-Enrichment Kit (StemCell Technologies) before staining for FACS-sorting.

Yu C.I., Becker C., Metang P., Marches F., Wang Y., Toshiyuki H., Banchereau J., Merad M, & Palucka K. (2014). Human CD141+ DCs induce CD4+ T cells to produce type 2 cytokines. Journal of immunology (Baltimore, Md. : 1950), 193(9), 4335-4343.

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Isolation and Enrichment of DCs

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PBMCs were isolated from leukapheresis products using Ficoll-Paque Plus density-gradient centrifugation. DCs were
enriched using human Pan-DC Pre-Enrichment Kit (StemCell Technologies) before FACS-sorting or use in co-culture.

Graham J.P., Authie P., Yu C.I., Zurawski S.M., Li X.H., Marches F., Flamar A.L., Acharya A., Banchereau J, & Palucka A.K. (2016). Targeting dendritic cells in humanized mice receiving adoptive T cells via monoclonal antibodies fused to Flu epitopes. Vaccine, 34(41), 4857-4865.

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Isolation and Sorting of Human Blood Dendritic Cells

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Human blood DCs were isolated from PBMCs using leukapheresis products. DCs were first enriched using human Pan-DC Pre-Enrichment Kit (StemCell Technologies) and then stained with fluorochrome-conjugated specific antibodies including Lineage cocktail 1-FITC (BD, dilution: 1:10), CD11c-V450 (B-ly6, BD, dilution: 1:50), CD1c/BDCA1-PerCp-Cy5.5 (L161, Biolegend, dilution 1:25), HLA-DR-APC-eFlour780 (LN3, eBioscience, dilution: 1:50), CD303/BDCA-2-PE (AC144, Mitenyi Biotec, dilution: 1:25) and CD141/BDCA3-APC (AD5-14H12, Mitenyi Biotec, dilution: 1:25). After washing with PBS, DCs were sorted as Lineage-HLA-DR+CD11c+ cells differentially expressing CD1c and CD141 using FACSAria II with Diva software (BD).

Banchereau R., Baldwin N., Cepika A.M., Athale S., Xue Y., Yu C.I., Metang P., Cheruku A., Berthier I., Gayet I., Wang Y., Ohouo M., Snipes L., Xu H., Obermoser G., Blankenship D., Oh S., Ramilo O., Chaussabel D., Banchereau J., Palucka K, & Pascual V. (2014). Transcriptional specialization of human dendritic cell subsets in response to microbial vaccines. Nature Communications, 5, 5283.

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Isolation and Sorting of Human Blood Dendritic Cells

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Human blood DCs were isolated from PBMCs using leukapheresis products. DCs were first enriched using human Pan-DC Pre-Enrichment Kit (StemCell Technologies) and then stained with fluorochrome-conjugated specific antibodies including Lineage cocktail 1-FITC (BD, dilution: 1:10), CD11c-V450 (B-ly6, BD, dilution: 1:50), CD1c/BDCA1-PerCpCy5.5 (L161, Biolegend, dilution 1:25), HLA-DR-APC-eFlour780 (LN3, eBioscience, dilution: 1:50), CD303/BDCA-2-PE (AC144, Mitenyi Biotec, dilution: 1:25) and CD141/BDCA3-APC (AD5-14H12, Mitenyi Biotec, dilution: 1:25). After washing with PBS, DCs were sorted as Lineage-HLA-DR+CD11c+ cells differentially expressing CD1c and CD141 using FACSAria II with Diva software (BD).

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Induction of Regulatory T Cells from Naïve CD4+ T Cells

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Fresh blood was obtained from 30 healthy volunteers, (age range from 34 to 62, median age is 46). Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized fresh blood by standard density gradient centrifugation with Ficoll-Paque Plus (GE Healthcare). Subsequently, Pan-DCs were obtained by negative selection using a Human Pan-DC pre-enrichment kit (Stem Cell Technologies). DCs were cultured in CellGenix GMP DC medium (CellGenix), and stimulated with LPS-EB Ultrapure (Sigma-Aldrich) and INFγ (Peprtotech); both at 100 ng/ml or not for 24 h, and then co-cultured with CD4+CD45RA+ naïve T cells in the anti-CD3 (eBioscience) 1 μg/ml for 4 days, and FOXP3 expression was detected by flow cytometry.

Zheng Y., Chen Z., Han Y., Han L., Zou X., Zhou B., Hu R., Hao J., Bai S., Xiao H., Li W.V., Bueker A., Ma Y., Xie G., Yang J., Chen S., Li H., Cao J, & Shen L. (2020). Immune suppressive landscape in the human esophageal squamous cell carcinoma microenvironment. Nature Communications, 11, 6268.

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