DCs were derived from bone marrow (BM) progenitor cells. In brief, femoral and tibial cells were harvested in DC culture medium (IMDM medium, 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 55 μM β-mercaptoethanol, 20 ng/mL GM-CSF, and 10 ng/mL IL-4) and seeded in 24-well plates at a density of 1 × 106 cells/ml/well. Culture medium was replaced with fresh medium after 3 days. On days 5–6, dislodged cells were used as BM-derived DCs. Splenic and mesenteric lymph node (MLN) DCs were isolated using magnetic CD11c beads according to the manufacturer’s instructions (Miltenyi Biotec) or sorted via flow cytometry. T cells from the lamina propria were isolated from colonic segments as has been previously described (David et al., 2017 (link)).
Cd11c magnetic beads
Manufactured by Miltenyi Biotec
Sourced in Germany, United States
CD11c magnetic beads are a lab equipment product designed for the isolation and enrichment of CD11c-positive cells. They consist of superparamagnetic iron oxide particles coated with antibodies specific to the CD11c surface antigen. These beads can be used in conjunction with a magnetic separation device to selectively capture and isolate CD11c-expressing cells from heterogeneous cell samples.
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Lab products found in correlation
Recombinant murine gm csf, by R&D Systems (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Rmil 4, by R&D Systems (4 mentions)
Lipopolysaccharides (lps), by Merck Group (3 mentions)
Rmil 1β, by R&D Systems (3 mentions)
Rmgm csf, by R&D Systems (3 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (3 mentions)
Gm csf, by Thermo Fisher Scientific (2 mentions)
Interleukin 4 (il 4), by R&D Systems (2 mentions)
Gm csf, by R&D Systems (2 mentions)
Rmgm csf, by Merck Group (2 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (2 mentions)
Macs column, by Miltenyi Biotec (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Rmtnf α, by R&D Systems (2 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by R&D Systems (1 mentions)
Spautin 1, by Merck Group (1 mentions)
Anti mouse igg microbeads, by Miltenyi Biotec (1 mentions)
30 protocols using cd11c magnetic beads
1
Isolation and Culture of Dendritic Cells
2
Isolation and Culture of Dendritic Cells
3
Dendritic Cell Transcriptome Analysis
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Dendritic cells (DCs) were separated from the other cells of the MLN of the PBS-immunized and experimental immunized mice using CD11c+ magnetic beads (Miltenyi Biotech). Total RNA was extracted from the DCs, and cDNA was prepared using a cDNA synthesis kit according to the manufacturer’s instructions. Transcript levels were determined by quantitative real-time PCR, using SYBR Green PCR Master Mix (Applied Biosystem) on StepOnePlus real time PCR system (Applied Biosystem). GAPDH levels were taken for normalization, fold changes were calculated using 2−ΔΔCt. In a separate experiment, CTB-T2544 and PBS immunized mice were euthanized by gradual carbon dioxide inhalation as described under Methods section after 12 days of three successful immunization and CD11cintMHCIIhiCD103+ cells were stained with fluorochrome-tagged antibody and evaluated using flow cytometer.
4
Isolation and Enrichment of Lung Dendritic Cells
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CD11c+ cells were isolated from single cell lung suspension using CD11c+ magnetic beads (Miltenyi Biotec, Auburn, CA, USA) according to manufacturer's instructions. Briefly, cells were washed in 1 ml of MACS buffer, incubated on ice with CD11c+ beads for 30 min. Thereafter, cell suspension was washed twice with MACS buffer and passed through a magnetic column. The CD11c+ cells were then collected by positive selection. Macrophage population was removed from the CD11c+ cells by overnight plastic adherence incubation. Later, cells were washed with RPMI and enriched for DCs by culturing for 7 days in complete medium in presence of GM-CSF (20 ng; Pepro Tech, Rocky Hill, NJ, USA). Cultures were fed every 48 h. The purity of DCs was >90% as enumerated by flow cytometry.
5
Isolation of mouse dendritic cells
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According to mice anatomy atlas, we collected axillary LNs, inguinal LNs, and other TDLNs. These LNs were minced with a scalpel blade and passed through a 100 μm nylon mesh. The suspension was collected, washed three times and re-suspended in PBS. Mouse DCs were isolated from LNs using CD11c magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany) for functional experiment on day 14. T lymphocytes from spleen of BALB/c or C57BL/6 mice were isolated using CD3 magnetic beads on day 14.
6
Isolation of CD11c+ Dendritic Cells
7
Isolation and Activation of Mouse Bone Marrow-Derived Dendritic Cells
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Six-week-old male C57BL/6J mice (bred at the Experimental Animal Centre, Second Military Medical University) were maintained in a pathogen-free animal facility for one week prior to use.
Mouse bone marrow DCs (BMDCs) were isolated from the femur bone marrow of C57/BL6J mice. After removing red blood cells by lysis, bone marrow cells were cultured in RPMI1640 media containing 10% Fetal Bovine Serum (FBS), 1000 U/ml GM-CSF (R&D Systems, Minneapolis, MN, USA) and 1000 U/ml IL-4 (R&D Systems, Minneapolis, MN, USA) overnight. The next day, non-adherent cells were removed by pipetting gently, and the adherent cells were cultured in the same medium for two more days. These cells were sorted using CD11c magnetic beads (Miltenyi, Bergisch Gladbach, North Rhine-Westphalia, Germany). CD11c+ cells were activated using LPS (Sigma-Aldrich, St. Louis, Missouri, USA) for 24 hours [40] .
Mouse bone marrow DCs (BMDCs) were isolated from the femur bone marrow of C57/BL6J mice. After removing red blood cells by lysis, bone marrow cells were cultured in RPMI1640 media containing 10% Fetal Bovine Serum (FBS), 1000 U/ml GM-CSF (R&D Systems, Minneapolis, MN, USA) and 1000 U/ml IL-4 (R&D Systems, Minneapolis, MN, USA) overnight. The next day, non-adherent cells were removed by pipetting gently, and the adherent cells were cultured in the same medium for two more days. These cells were sorted using CD11c magnetic beads (Miltenyi, Bergisch Gladbach, North Rhine-Westphalia, Germany). CD11c+ cells were activated using LPS (Sigma-Aldrich, St. Louis, Missouri, USA) for 24 hours [40] .
8
Splenic DC Isolation and Ischemia Simulation
9
Isolation of Mouse B Cells, DCs, and LMΦ
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B cells were isolated from mouse spleens using anti-CD19 magnetic beads according to the manufacturer’s instructions. In brief, the cells were incubated with anti-CD19 magnetic beads for 15 min on ice and washed with 1 ml of MACS buffer (solution containing phosphate-buffered saline [PBS], pH 7.2, 0.5% bovine serum albumin [BSA], and 2 mM EDTA) (Miltenyi Biotec, Bergisch Gladbach, Germany). The cellular suspensions collected were washed twice in MACS buffer and passed through a magnetic column. Then, CD19+ B cells were isolated by positive selection, washed, resuspended in complete RPMI 1640 medium, and counted before coculture with DCs or lung macrophages (LMφ), as described below.
From the mononuclear cells isolated from the mouse lungs, spleen, and bone marrow, CD11c+ DCs were isolated using CD11c magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany). CD64+ LMφ were isolated using a CD64 primary antibody (clone X54-5/7.1; BioLegend, CA, USA) and anti-mouse IgG microbeads (Miltenyi Biotec), as described for B cell isolation.
From the mononuclear cells isolated from the mouse lungs, spleen, and bone marrow, CD11c+ DCs were isolated using CD11c magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany). CD64+ LMφ were isolated using a CD64 primary antibody (clone X54-5/7.1; BioLegend, CA, USA) and anti-mouse IgG microbeads (Miltenyi Biotec), as described for B cell isolation.
10
Isolation and Activation of Human and Murine Dendritic Cells
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Human myeloid DCs were isolated from peripheral blood. After Ficoll-Hypaque (PAA, GE, United Kingdom) separation, lymphocytes were sorted using CD14 magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany). CD14 cells were cultured in RPMI 1640 containing 10% FBS, 50 ng/mL GM-CSF (R&D, Minnesota, USA), and 10 ng/mL IL-4 (R&D Systems, Minneapolis, MN, USA) overnight. The following day, non-adherent cells were removed by gentle pipetting and adherent cells were cultured in the same medium for two additional days. DCs were activated using lipopolysaccharides (LPS) (Sigma-Aldrich, St. Louis, MO, USA) for 24h.
Murine DCs were isolated from tumor-draining lymph nodes. After removal of red blood cells through lysis, DCs were cultured overnight in RPMI 1640 containing 10% FBS, 1000 U/mL GM-CSF (R&D Systems), and 1000 U/mL IL-4 (R&D Systems). The following day, non-adherent cells were removed by gentle pipetting and adherent cells were cultured in the same medium for two additional days. The cells were sorted using CD11c magnetic beads (Miltenyi Biotec). CD11c+cells were then activated with LPS (Sigma-Aldrich) for 24 h.
Murine DCs were isolated from tumor-draining lymph nodes. After removal of red blood cells through lysis, DCs were cultured overnight in RPMI 1640 containing 10% FBS, 1000 U/mL GM-CSF (R&D Systems), and 1000 U/mL IL-4 (R&D Systems). The following day, non-adherent cells were removed by gentle pipetting and adherent cells were cultured in the same medium for two additional days. The cells were sorted using CD11c magnetic beads (Miltenyi Biotec). CD11c+cells were then activated with LPS (Sigma-Aldrich) for 24 h.
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