To rapidly isolate RNA from 12.5 Gy and non-irradiated tissue for sequencing, we developed a technique of rapidly isolating pulmonary epithelia based on the expression of the epithelial cell-adhesion molecule (EpCam) CD326 (Fig. 1 ). To isolate CD326+ epithelium from whole lung-tissue digest, cells were pelleted and resuspended in microbead buffer (PBS, 0.5% BSA, and 2 mM EDTA) plus CD45 magnetic microbeads (Miltenyi Biotech #130 052 301, Auburn, CA) per manufacturer’s instructions to remove the CD45+ fraction of cells from the lung homogenate. This CD45-depleted population of cells was then recounted, pelleted, and resuspended in bead buffer plus CD326 microbeads (Miltenyi Biotech #130 105 958). Again, cells were washed in buffer solution, pelleted, and resuspended in 500 μl fresh buffer prior to a second magnetic separation using a Miltenyi LS column. Unbound CD326 negative cells were washed from the column, and CD326+ cells trapped within the column were flushed from the column and collected. Cells were pelleted, washed, and transferred into 600μl RLT Lysis buffer (Qaigen, Hilden, Germany) and stored at −80°C until processing for RNA isolation. All RNA isolation, quality-control analysis, and low-input RNA sequencing procedures were performed by the University of Rochester Genomics Research Center.
Cd45 magnetic microbeads
Manufactured by Miltenyi Biotec
Sourced in Germany
CD45 magnetic microbeads are a laboratory tool used for the isolation and enrichment of CD45-positive cells from various sample types. They consist of superparamagnetic iron oxide particles coated with antibodies specific to the CD45 cell surface antigen. These beads enable the rapid and efficient separation of CD45-expressing cells, such as leukocytes, from complex cell mixtures using a magnetic field. The core function of CD45 magnetic microbeads is to facilitate the isolation and purification of CD45-positive cells for further analysis or downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Collagenase, by Worthington (2 mentions)
Ms column, by Miltenyi Biotec (2 mentions)
Dmem f12, by Corning (2 mentions)
Nylon cell strainer, by Corning (2 mentions)
Cd326 microbeads, by Miltenyi Biotec (1 mentions)
Ls column, by Miltenyi Biotec (1 mentions)
Facsaria fusion cell sorter, by BD (1 mentions)
Gentlemacs c tube, by Miltenyi Biotec (1 mentions)
Automacs cell separator, by Miltenyi Biotec (1 mentions)
Gentlemacs dissociator, by Miltenyi Biotec (1 mentions)
Tumor dissociation kit, by Miltenyi Biotec (1 mentions)
6 protocols using cd45 magnetic microbeads
1
Rapid Isolation of Epithelial RNA
2
Single-Cell Transcriptomics of Skin Tissue
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Single-cell gene expression profiles were generated using the 10x Genomics Chromium (3′RNA-seq) platform. Briefly, skin tissues were digested using 20 mg/mL Collagenase (Worthington, Cat#LS004196) dissolved in DMEM/F12 (Corning, VWR# 45000-350) media. Digested tissues were passed through 100 and 70 µm nylon cell strainers (Corning, VWR# 10054-458, 89508-344). Single-cell suspensions were washed by MACS buffer, incubated with CD45+ magnetic microbeads (Miltenyi Biotec, Cat# 130-052-301), and passed through MS Columns (Miltenyi Biotec, 130-042-201) following the protocol provided in the kit. MACS buffer consists of 1xPBS with 2 mM EDTA (VWR, Cat#97062-836) and 0.5% BSA (VWR, Cat#EM2930). Single-cell suspensions were kept in PBS with 0.04% BSA for sequencing. Sequencing results were processed by Cell Ranger. QC was analyzed by Seurat with percent.mt<10; nFeature_RNA > 200, nFeature_RNA < 4000, (WT_UV nFeatures_RNA < 6000 due to deeper illumina sequencing). Data integration was accomplished by Seurat4 SCTintegration. Pseudotime analysis was performed by Monocle 3. CellChat signaling network analysis was performed by CellChat 1.5.0. Code can be found in figshare.
3
Single-cell RNA-seq of Skin Tissue
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Single cell gene expression profiles were generated using the 10x Genomics Chromium (3’RNA-seq) platform. Briefly, skin tissues were digested using 20mg/mL Collagenase (Worthington, Cat#LS004196) dissolved in DMEM/F12 (Corning, VWR# 45000–350) media. Digested tissues were passed through 100 and 70 um nylon cell strainers (Corning, VWR# 10054–458, 89508–344). Single cell suspensions were washed by MACS buffer, incubated with CD45+ magnetic microbeads (Miltenyi Biotec, Cat# 130–052-301) and passed through MS Columns (Miltenyi Biotec, 130–042-201) following the protocol provided in the kit. MACS buffer consists of 1xPBS with 2mM EDTA (VWR, Cat#97062–836) and 0.5% BSA (VWR, Cat#EM2930). Single cell suspensions were kept in PBS with 0.04% BSA for sequencing. Sequencing results were processed by Cell Ranger. QC was analyzed by Seurat with percent.mt<10; nFeature_RNA>200, nFeature_RNA<4000, (WT_UV nFeatures_RNA<6000 due to deeper illumina sequencing). Data integration was accomplished by Seurat4 SCTintegration. Pseudotime analysis was performed by Monocle 3. CellChat signaling network analysis was performed by CellChat 1.5.0. Code can be found in figshare.
4
Isolation of CD45- Cells from Human FL
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Single human FL cells were separated by Ficoll density gradient centrifugation at 400 g for 30 min. The mononuclear cells were collected and incubated with CD45 magnetic microbeads (Catalog No. 130-045-801, Miltenyi Biotec, Bergisch Gladbach, Germany) for 30 min at 4 °C in the dark, according to the manufacturer’s protocol. The CD45− cells were collected using a magnetic-activated cell sorting magnetic bead system. The isolated CD45− cells were suspended in staining buffer (PBS supplemented with 2 mM EDTA and 0.5% BSA) and blocked with 0.4% human AB serum for 10 min. The cells were stained with PE-conjugated GPA, APC-conjugated α4-integrin, and FITC-conjugated band 3 at room temperature in the dark. After 15 min, the cells were washed with staining buffer by centrifugation at 300 g for 5 min at 4 °C and then resuspended in staining buffer with 7-AAD. The cells were finally sorted on a BD FACSAria fusion cell sorter (BD Biosciences).
5
Isolation of Tumor-Infiltrating Leukocytes
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Single cell suspensions were prepared from the spleens, MLNs, and ILNs by mechanical dissociation as previously described (26 (link)). Tumors were processed using a previously described method, with modification (27 (link)). Briefly, dissected tumor tissues were subjected to mechanical and enzymatic digestion in gentleMACS C-tubes (Miltenyi Biotec, Germany) using a tumor dissociation kit (Miltenyi Biotec) and the GentleMACS dissociator (Miltenyi Biotec), according to manufacturer’s instructions. Tumor-infiltrating leukocytes (TILs) were subsequently purified from tumor cell suspensions using magnetic CD45+ microbeads and the autoMACS cell separator, according to the manufacturer’s protocol (Miltenyi Biotec).
6
Co-culture of hMSCs and Macrophages
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For co-culture experiments, monocytes/macrophages were cultivated four days on suspension plates (Sarstedt, Nürnbrecht (D)). After that time, hMSCs in a ratio of one (hMSCs) to four (macrophages) in passage 2 were added onto the monocytes/macrophages in fresh macrophage culture medium. Co-culture studies were performed for up to three days. For gene expression analysis, macrophages were separated after co-cultivation via magnetic CD45 MicroBeads (Miltenyi Biotec, Bergisch Gladbach (D)) according to the manufacturer’s protocol.
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