Thymi from wild-type mice were dissociated using collagenase (Sigma-Aldrich.) digestion for 1 h at 37°C. Some sample was reserved for RNA isolation (“whole thymocyte” samples), and the remainder was used for DC enrichment using MACS CD11c microbeads according to manufacturer’s instructions (Miltenyi), followed by flow cytometry to reach a final purity >85% CD11c+ F4/80− population (“thymic DC” samples). The thymus sample after removal of CD11c+ cells was also used for RNA isolation (“CD11c depleted” samples). Cell samples were lysed in Trizol (Life Technologies), and total RNA prepared using the RNAeasy Kit (Qiagen) as per manufacturer’s instructions. Reverse transcription was performed using the Quatitect RT Kit (Qiagen) and cDNA was utilized to run qPCR reactions using Taqman probes along with TaqMan Real-Time PCR master mix (Life Technologies). PrimeTime probes, (Mm.PT.58.11478202 (IL-2) and Mm.PT.39a.1 (GAPDH)), were synthesized by IDT. All reactions were run on an Applied Biosystems 7300 RT PCR machine. IL-2 expression data were normalized to GAPDH and expressed as fold increase over the background values from Il2−/− bone marrow-derived DCs using ΔΔCt values. (For Il2−/− DCs, no IL2 signal was observed after 40 cycles of PCR, therefore values reported are upper estimates (indicated by grey shading), and were used to determine the lower limit of detection of the assay.
Macs cd11c microbeads
Manufactured by Miltenyi Biotec
Sourced in Germany
MACS CD11c microbeads are a magnetic labeling reagent used for the isolation of CD11c-positive cells from single-cell suspensions. They allow for the separation and enrichment of dendritic cells or other CD11c-expressing cell types.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (5 mentions)
Collagenase, by Merck Group (2 mentions)
Taqman real time pcr master mix, by Thermo Fisher Scientific (2 mentions)
Rnaeasy kit, by Qiagen (2 mentions)
Quatitect rt kit, by Qiagen (2 mentions)
7300 rt pcr machine, by Thermo Fisher Scientific (2 mentions)
Taqman probe, by Thermo Fisher Scientific (2 mentions)
Glutamine, by Sartorius (1 mentions)
Penicillin, by Sartorius (1 mentions)
Streptomycin, by Sartorius (1 mentions)
Hepes, by Sartorius (1 mentions)
Sodium pyruvate, by Sartorius (1 mentions)
Rpmi 1640 medium, by Sartorius (1 mentions)
Ova257 264, by InvivoGen (1 mentions)
β mercaptoethanol, by Sartorius (1 mentions)
Cd8a t cell isolation kit, by Miltenyi Biotec (1 mentions)
Automacs pro, by Miltenyi Biotec (1 mentions)
Aria 2 cell sorter, by BD (1 mentions)
Cd4 t cell isolation kit, by Miltenyi Biotec (1 mentions)
Granulocyte macrophage colony stimulating factor gm csf, by Thermo Fisher Scientific (1 mentions)
8 protocols using macs cd11c microbeads
1
Isolation and qPCR Analysis of Thymic DCs
2
OT-I CD8+ T-cell Activation by OVA Peptide
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CD8+ T-cells were purified (>95%) from a cell suspension harvested from crushed spleens of OT-I mice, using a CD8a+ T Cell Isolation Kit and magnetic-associated cell sorting (MACS), according to the manufacturer’s instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). Similarly, DCs were purified (>85%) from spleens of C57BL/6 mice, using MACS CD11c microbeads (Miltenyi Biotec). T-cells and DCs were cultured immediately following isolation in a ratio of 3:1, respectively, with 1 µg/ml of ovalbumin peptide (OVA257-264, InvivoGen, San Diego, CA, USA) in a RPMI 1640 medium w/o phenol red, supplemented with 10% serum, 100 U/ml of penicillin, 100 mg/ml of streptomycin, 2 mM glutamine, 10 mM HEPES, 1 mM sodium pyruvate, and 50 mM β-mercaptoethanol (Biological Industries, Beit Haemek, Israel).
3
Isolation and qPCR Analysis of Thymic DCs
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Thymi from wild-type mice were dissociated using collagenase (Sigma-Aldrich.) digestion for 1 h at 37°C. Some sample was reserved for RNA isolation (“whole thymocyte” samples), and the remainder was used for DC enrichment using MACS CD11c microbeads according to manufacturer’s instructions (Miltenyi), followed by flow cytometry to reach a final purity >85% CD11c+ F4/80− population (“thymic DC” samples). The thymus sample after removal of CD11c+ cells was also used for RNA isolation (“CD11c depleted” samples). Cell samples were lysed in Trizol (Life Technologies), and total RNA prepared using the RNAeasy Kit (Qiagen) as per manufacturer’s instructions. Reverse transcription was performed using the Quatitect RT Kit (Qiagen) and cDNA was utilized to run qPCR reactions using Taqman probes along with TaqMan Real-Time PCR master mix (Life Technologies). PrimeTime probes, (Mm.PT.58.11478202 (IL-2) and Mm.PT.39a.1 (GAPDH)), were synthesized by IDT. All reactions were run on an Applied Biosystems 7300 RT PCR machine. IL-2 expression data were normalized to GAPDH and expressed as fold increase over the background values from Il2−/− bone marrow-derived DCs using ΔΔCt values. (For Il2−/− DCs, no IL2 signal was observed after 40 cycles of PCR, therefore values reported are upper estimates (indicated by grey shading), and were used to determine the lower limit of detection of the assay.
4
Isolation of Splenocytes, DCs and CD4+ T Cells
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Single cell suspensions of splenocytes were generated by rinsing spleens with erythrocyte lysis buffer and washing with PBS supplemented with 2% FCS and 2 mM EDTA. For the isolation of CD11c+ DCs, splenocytes were separated from contaminating superparamagnetic splenic red pulp cells (32 (link)) by using the AutoMACS Pro (Miltenyi Biotec, Bergisch Gladbach, Germany) before using MACS CD11c Microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s recommendations. CD4+ T cells were isolated from splenocytes using the CD4+ T cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s protocol, followed by anti-CD4 staining and cell sorting using an Aria II Cell Sorter (BD Biosciences, Heidelberg, Germany).
5
Isolation and Generation of Murine Dendritic Cells
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To isolate liver DCs, CD11c+ cells were positively sorted from liver MNCs using MACS CD11c microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). For the generation of mouse DCs, mouse bone marrow cells were obtained by flushing the humerus and femur bones with PBS containing 2% FBS (Gibco, Grand Island, NY, USA). DCs were cultured in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF; 10 ng/mL) and interleukin (IL)-4 (5 ng/mL) (both from PeproTech, Rocky Hill, NJ, USA) for 5 days to induce immature DCs (immature DC induction phase). The cells were further incubated with GM-CSF and tumor necrosis factor (TNF)-α (50 ng/mL; PeproTech) on type I collagen-coated plates for 3 more days to induce mature DCs (mature DC induction phase). LPS (0.1 μg/mL) was added on day 7 to further promote DC maturation.
6
Quantification of MCMV and CD70 in DCs
7
Isolation and Quantification of Immune Cells
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Cells from BAL, lung, and dMLNs were harvested as described above. CD11c+ cells were sorted by using CD11c MACs microbeads (Miltenyi Biotech). Sorted cells were spun down and put into 0.5 mL TRIzol (Invitrogen) for RNA extraction. cDNA was synthesized using High Capacity Reverse Transcription kit (Applied Biosystems, Foster City, CA). For real-time PCR, all primer and probe mixes and TaqMan fast master mixes were purchased from Applied Biosystems. For genes of interest, mRNA expression was quantified by using ABI 7500 fast real-time PCR system, according to the manufacturer's instructions. The mRNA relative expression level of target gene was normalized to housekeeping gene 18s.
8
Isolation and Activation of Splenic Dendritic Cells
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The spleens of septic mice were harvested and washed with phosphate-buffered saline (PBS). Mononuclear cells were separated from the splenic tissue using 40 μm meshes and mononuclear cells were obtained through density gradient centrifugation using NycoPrep (Axis-shield Co., Norway). Splenic DCs were obtained using CD11c MACS microbeads (Miltenyi Biotech, Bergisch Gladbach, Germany) while T lymphocytes were isolated using CD4 MACS microbeads (Miltenyi Biotech). Immature CD11c+ DCs were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS) at 37 °C and 5% CO2. In vitro sepsis models, the CD11c+ DCs were treated with 1000 ng/mL LPS (Escherichia coli strain, Sigma).
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