Cd11c positive selection kit

Manufactured by STEMCELL

Sourced in Canada

The CD11c Positive Selection Kit is a laboratory tool used to isolate CD11c-positive cells from a mixed cell population. It allows for the enrichment and purification of these cells for further analysis or research purposes.

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Lab products found in correlation

Gm csf, by R&D Systems (2 mentions) Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions) Cd4 negative selection, by STEMCELL (1 mentions) Facsaria 2, by BD (1 mentions) Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions) Streptomycin, by Merck Group (1 mentions) Recombinant mouse gm csf, by BioLegend (1 mentions) Cd8 t cell negative isolation kit, by STEMCELL (1 mentions) Cytometric bead array assay, by BD (1 mentions) Collagenase 8, by Merck Group (1 mentions) Dnase 1, by Merck Group (1 mentions) Cba assay, by BD (1 mentions) Cyclophilin a, by Cell Signaling Technology (1 mentions) Clarity max western ecl substrate, by Bio-Rad (1 mentions) Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions) Keyhole limpet hemocyanin (klh), by Merck Group (1 mentions) Poly 1 c, by InvivoGen (1 mentions) Cd4 positive selection kit, by STEMCELL (1 mentions) Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions) Recombinant mouse gm csf, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

EasySep Mouse CD11c Positive Selection Kit (21 citations) EasySep Mouse CD11c Positive Selection Kit II (14 citations) Mouse CD11c Positive Selection Kit (4 citations) CD11c positive selection magnetic bead kit (2 citations) Mouse CD11c Positive Selection Kit II (2 citations) EasysepTM CD11c positive selection kit (2 citations)

11 protocols using cd11c positive selection kit

Dendritic Cell-Mediated T Cell Proliferation

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Bone marrow-derived dendritic cells (BMDCs) were generated by culturing bone marrow cell suspensions in 20ng/ml recombinant GM-CSF (Peptrotech) for 10 days. For necroptosis assays, BMDCs were treated with 0.1 μM Smac mimetic (ChemieTek) and 10 μM zVAD (Enzo). For apoptosis assays, BMDCs were treated with cyclohexmide (0.5 μg/ml), TNFα (10 ng/ml), IFNγ (10 ng/ml) or with FasL and control vesicles purified from N2-mFasL and N2-neo cell supernatant (diluted 1/40), as previously described (24 (link)). Splenic DC were isolated from mice following treatment of the Flt3L producing melanoma line B16, using a CD11c positive selection kit (Stemcell Technologies).
To examine T cell proliferation, purified CD11c⁺ splenic DC from CD11cCre and Ripk1^{DC KO} mice were incubated with OVA_323–339 or control OVA_257–264 peptide for 1 h. CD4⁺ T cells were isolated from spleens of OT-II mice using CD4 positive selection beads (Invitrogen). Isolated CD4⁺ cells were labeled with 0.5 μM CFSE (Invitrogen) and incubated with splenic DCs for 72 h. CFSE staining was examined in viable CD4⁺ cells by flow cytometry.

O’Donnell J.A., Lehman J., Roderick J.E., Martinez-Marin D., Zelic M., Doran C., Hermance N., Lyle S., Pasparakis M., Fitzgerald K.A., Marshak-Rothstein A, & Kelliher M.A. (2017). Dendritic cell RIPK1 maintains immune homeostasis by preventing inflammation and autoimmunity. Journal of immunology (Baltimore, Md. : 1950), 200(2), 737-748.

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Isolation and Culturing of Murine CD4+ T Cells

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CD4⁺ T cells from mouse LNs and spleens were isolated using CD4⁺-negative selection (Stemcell Technologies, Cambridge, MA), and were cultured as previously described (1). Briefly CD4⁺CD25⁻Foxp3GFP⁻ WT or Ltα^−/− cells with >98% purity were sorted using a FACS Aria II (BD Biosciences). The sorted GFP⁻ naïve CD4 T cells (nCD4) (5 × 10⁴) were then co-cultured with T cell-depleted, 800 rad-irradiated C57BL/6 splenocytes as stimulator cells (5 × 10⁴) in U-bottom 96-well plates for 5 days at 37 °C in 5% CO₂, with IL-2 (20 ng/mL; eBioscience, San Diego, CA), anti-CD3ɛ mAb (1 μg/mL, clone 145-2C11; eBioscience) for activated T cells (aCD4); and human TGF-β1 (10 ng/mL; eBioscience) for induced regulatory T cells (iTreg). Cells were cultured in RPMI 1640 supplemented with 10% FBS, 1 mM sodium pyruvate, 2 mM l-glutamine, 100 IU/mL penicillin, 100 μg /mL streptomycin, non-essential amino acids and 2 × 10⁻⁵ M 2-ME (Sigma-Aldrich). BMDCs were generated as described^{51 (link)}. Briefly, bone marrow (BM) cells of wild-type mice were treated with 10 ng/mL GM-CSF (R&D Systems, Minneapolis, MN) for 10 days in Petri dishes, and the loosely attached cells were collected and CD11c⁺ DC were purified by CD11c-positive selection kit (Stemcell Technologies).

Piao W., Xiong Y., Famulski K., Brinkman C.C., Li L., Toney N., Wagner C., Saxena V., Simon T, & Bromberg J.S. (2018). Regulation of T cell afferent lymphatic migration by targeting LTβR-mediated non-classical NFκB signaling. Nature Communications, 9, 3020.

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Priming Antigen-Specific CD8+ T Cells

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Single-cell suspensions of bone marrow (BM) cells were collected from tibias and femurs of C57BL/6 mice. The BM cells were placed in 10cm dish and cultured with complete RPMI 1640 medium containing 20 ng/mL recombinant mouse GM-CSF (BioLegend). Fresh medium with was added into the culture on days 3 and 6. The BMDCs were harvested Day 7. CD8⁺ T cells were isolated from lymph nodes and spleens of OT-1 transgenic mice with a negative CD8⁺ T cell isolation kit (Stemcell). MC38-OVA cells pretreated with 200 nM 6-thio-dG for 4 h. Then the drug was washed out, tumor cells were continued to culture for 72 h and were harvested on the same day as BMDC harvest. Then MC38-OVA cells were co-cultured with BMDC for overnight. Supernatant was collected for IFN-β ELISA test (PBL). BMDCs were sorted with CD11c positive selection kit (Stemcell) and co-cultured with OT-1 CD8⁺ T cells for 48 h. Supernatants were collected and IFN-γ was measured by cytometric bead array assay (BD Biosciences).

Mender I., Zhang A., Ren Z., Han C., Deng Y., Siteni S., Li H., Zhu J., Vemula A., Shay J.W, & Fu Y.X. (2020). Telomere Stress Potentiates STING-Dependent Anti-Tumor Immunity. Cancer cell, 38(3), 400-411.e6.

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Isolation and Culture of Murine Bone Marrow-Derived Dendritic Cells

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Female C57BL/6 mice (n = 3-5/ experiment) were euthanized and bone marrow was collected from the tibia and femur under aseptic conditions. BMDC were prepared as described previously with slight modifications [26 (link)]. At day 7 of culture, all cells were pooled and enriched using CD11c positive selection kit (Stem Cell Technologies, Vancouver). Following enrichment (purity > 85% by flow cytometry), BMDC were cultured in 96 well plates and treated with miR-155 mimic and/or inhibitor as described below.

Gupta R., Arkatkar T., Keck J., Koundinya G.K., Castillo K., Hobel S., Chambers J.P., Yu J.J., Guentzel M.N., Aigner A., Christenson L.K, & Arulanandam B.P. (2016). Antigen specific immune response in Chlamydia muridarum genital infection is dependent on murine microRNAs-155 and -182. Oncotarget, 7(40), 64726-64742.

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CD11c+ DC Activation Assay

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B16-EGFR-SIY bearing mice were treated with 25 μg of anti-EGFR, or anti-EGFR-IFNβ intratumoral injection on days 14 and 17. Four days later, draining lymph node was digested with 1mg/ml of collagenase VIII (Sigma) and 200ug/ml DNase I (Sigma) at 37°C for 15 minutes. DCs were purified by CD11c positive selection Kit (Stemcell). Approximately 1×10⁵ DCs were mixed together with purified 2×10⁵ 2C T cells with or without 5μg/mL of SIY peptide to restimulate the T cells. Two days later, the supernatants were collected, and IFNγ was measured by CBA assay (BD Bioscience).

Yang X., Zhang X., Fu M.L., Weichselbaum R.R., Gajewski T.F., Guo Y, & Fu Y.X. (2014). Targeting the tumor microenvironment with interferon-β bridges innate and adaptive immune responses. Cancer cell, 25(1), 37-48.

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Immunoblotting Analysis of STING Pathway

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BMDC and MC38 treatment were same as “In vitro co-culture of bone marrow dendritic cells”. 6 h after co-culture, DC was isolated with CD11c positive selection kit (Stemcell). Protein sample preparation and immunoblot procedures were performed as previously described (Liu et al., 2019 ). Proteins were detected with rabbit monoclonal antibodies for pSTING (Cell signaling, 72971), STING (Cell signaling, 50494), pTBK1 (Cell signaling, 5483), TBK1 (Cell signaling, 3504). Protein loading was determined with antibodies against with Cyclophilin A (Cell signaling, 2175). Anti-rabbit was used for secondary antibody (Cell signaling, 7074). X-ray film (GeneMate, F-9024–8X10) was used to develop the membranes. Clarity Max Western ECL Substrate (Biorad, 1705062) or Supersignal West PicoPlus Chemiluminescent Substrate (Thermoscientific, 34577) was used for chemiluminescent western blot.

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T Cell Purification and Adoptive Transfer

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After removing B cells (using anti-mouse Ig) and other APCs (I-A^g7 major histocompatibility complex [MHC] class II⁺) using anti-I-A^g7 (10.2.16) mAb and magnetic bead separation, CD4⁺ or CD8⁺ T cells were purified by magnetic bead–based negative selection using the anti-CD8 (T1B105) or anti-CD4 (GK1.5) mAb, respectively. The purity of the isolated T cells used for adoptive transfer experiments was routinely ≥90%, analyzed by flow cytometry. Dendritic cells (DCs) were purified by CD11c-positive selection kit (Stem Cell Technology).

Tan Q., Majewska-Szczepanik M., Zhang X., Szczepanik M., Zhou Z., Wong F.S, & Wen L. (2014). IRAK-M Deficiency Promotes the Development of Type 1 Diabetes in NOD Mice. Diabetes, 63(8), 2761-2775.

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T Cell Proliferation Assay with KLH-Loaded DCs

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NOD mice were injected intraperitoneally with keyhole limpet hemocyanin (KLH; Sigma), 200 μg/mouse as a foreign antigen, and were emulsified in Alum (Pierce). Mice were killed 7 days after immunization, and CD4⁺ and CD8⁺ T cells were isolated from the splenocytes by magnetic bead–based negative selection. DCs were purified from IRAK-M^−/− and WT NOD mice by a CD11c positive selection kit (Stemcell Technologies). DCs were or were not loaded with KLH (Sigma) 50 μg/mL for 5 h in complete medium at 37°C. Antigen-loaded DCs were then washed with PBS twice before coculturing with T cells. CD4⁺ and CD8⁺ T cells from immunized mice were cocultured with DCs from IRAK-M^−/− NOD or WT NOD mice, with or without KLH, at a 2:1 ratio in complete medium. ³H-thymidine was added during the last 16 h of a 5-day culture, to determine antigen-specific T-cell proliferation responses by ³H-thymidine incorporation. In DC vaccine experiments, purified DCs from immunized mice were preloaded with KLH, as described above, and 10⁶ cells were injected intravenously into WT NOD mice. The mice were killed 7 days after DC vaccination, and splenocytes were harvested and placed 10⁵/well with KLH (1 μg/mL) in complete medium. Proliferative responses were determined by ³H-thymidine incorporation. ³H-thymidine was added during the last 16 h of a 5-day culture.

Tan Q., Majewska-Szczepanik M., Zhang X., Szczepanik M., Zhou Z., Wong F.S, & Wen L. (2014). IRAK-M Deficiency Promotes the Development of Type 1 Diabetes in NOD Mice. Diabetes, 63(8), 2761-2775.

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Isolation and Polarization of Mouse Dendritic Cells and Macrophages

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For bone marrow-derived dendritic cells (BMDCs) preparation, bone marrow from 6 to 8-week-old C57BL/6 mice was cultured in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco), recombinant mouse GM-CSF (50 ng/mL; Peprotech), and IL-4 (20 ng/mL; Peprotech). Half medium was replaced by fresh medium with GM-CSF and IL-4 at day 3, and used at day 6 as immature DCs. Immature DCs were stimulated with TLR agonists LPS (50 ng/mL; Sigma-Aldrich) or poly I:C (20 ng/mL; InvivoGen) for 8 h to generate mature DCs.
For bone marrow-derived macrophages (BMDMs) preparation, bone marrow from 6 to 8-week-old C57BL/6 mice was cultured in DMEM medium (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco), recombinant mouse M-CSF (50 ng/mL; Peprotech). All supernate was replaced by fresh medium with M-CSF at day 3. BMDMs were then stimulated with LPS (50 ng/mL; Sigma-Aldrich) for M1 polarization, or IL-4 (20 ng/mL; Peprotech) for M2 polarization for 24 h at day 6.
CD11c⁺ DCs were isolated from the spleen using CD11c positive selection kit (STEMCELL Technologies), and CD4⁺ T cells were isolated from the lymph nodes using CD4 positive selection kit (STEMCELL Technologies).

Wang J., Wang J., Hong W., Zhang L., Song L., Shi Q., Shao Y., Hao G., Fang C., Qiu Y., Yang L., Yang Z., Wang J., Cao J., Yang B., He Q, & Weng Q. (2021). Optineurin modulates the maturation of dendritic cells to regulate autoimmunity through JAK2-STAT3 signaling. Nature Communications, 12, 6198.

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Th17 Cells and APC Interaction

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CD4⁺CCR6⁺CXCR3⁻ T cells (Th17 cells) were isolated by cell sorting and CD11C⁺ APCs were isolated by CD11C positive selection kit (STEMCELL, Vancouver, Canada). Then, sorted CD4⁺CCR6⁺CXCR3⁻ T cells and CD11C⁺ APCs were co-cultured at a ratio of 2:1 with or without anti-GM-CSF (10 g/mL, R&D Systems) for 24 h. The supernatants were used to measure IL-23 by ELISA kit (Invitrogen, Carlsbad, CA, USA).

Li H., Zhu L., Wang R., Xie L., Ren J., Ma S., Zhang W., Liu X., Huang Z., Chen B., Li Z., Feng H., Liu G.H., Wang S., Qu J, & Su W. (2021). Aging weakens Th17 cell pathogenicity and ameliorates experimental autoimmune uveitis in mice. Protein & Cell, 13(6), 422-445.

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