A dissociated single-cell suspension was passed through a 30-µm nylon mesh (Pre-Separation Filters; Miltenyi Biotec #130-041-407) to remove cell clumps that could clog the column. After counting cell numbers, an appropriate number of cells was pipetted, which was defined as pre-separation interstitial cells. The remaining cells were pelleted and resuspended in 80 µl of MACS buffer (PBS pH 7.2, 0.5% BSA and 2 mM EDTA) and 20 µl anti-CD45 MicroBeads (Miltenyi Biotec #130-052-301) or 20 µl anti-F4/80 MicroBeads (Miltenyi Biotec #130-110-443) per 107 total cells. The mixture was incubated in the dark at 4 °C for 15 min, washed, and resuspended in MACS buffer. Cell suspension was applied onto the pre-balanced LS column (Miltenyi Biotec #130–097-679) that was placed in the magnetic Separator. Then the LS column was removed from the separator and the magnetically labeled cells were immediately flushed out, which were defined as the CD45+ or F4/80+ fraction. After incubation with anti-CD45 MicroBeads, unlabeled cells which passed through the LS column and LD column (Miltenyi Biotec #130-042-901) were defined as the CD45- fraction. Each fraction was seeded in 24-well plates with or without glass coverslips and maintained in a humidified atmosphere (5% CO2, 95% air) at 37 °C for 3 days or 6 days in culture media (10% FBS in RPMI-1640).
Anti cd45 microbeads
Manufactured by Miltenyi Biotec
Sourced in Germany
Anti-CD45 microbeads are a laboratory reagent used for the isolation and purification of CD45-positive cells from complex biological samples. These microbeads are coated with antibodies specifically targeting the CD45 surface antigen, which is expressed on the majority of leukocytes. The core function of these microbeads is to facilitate the magnetic separation and enrichment of CD45-positive cells, allowing for their further analysis or downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Ls column, by Miltenyi Biotec (5 mentions)
Anti cd31 microbead, by Miltenyi Biotec (5 mentions)
Anti pe microbeads, by Miltenyi Biotec (4 mentions)
Dnase 1, by Merck Group (4 mentions)
Dnase 1, by Roche (4 mentions)
Collagenase dispase, by Roche (3 mentions)
Automacs, by Miltenyi Biotec (2 mentions)
Anti cd90.2 microbeads, by Miltenyi Biotec (2 mentions)
Octomacs separator, by Miltenyi Biotec (2 mentions)
Collagenase type 4, by Worthington (2 mentions)
Anti cd326 microbeads, by Miltenyi Biotec (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (2 mentions)
Moflo xdp, by Beckman Coulter (2 mentions)
Quantitect mm actb 1sg primer assay, by Qiagen (2 mentions)
Gentlemacs octo dissociator, by Miltenyi Biotec (2 mentions)
Mesencult expansion kit, by STEMCELL (2 mentions)
Collagenase type 2, by Worthington (2 mentions)
Large cell column, by Miltenyi Biotec (2 mentions)
Trypsin, by Thermo Fisher Scientific (2 mentions)
47 protocols using anti cd45 microbeads
1
Isolation and Culture of Immune Cell Subsets
2
Single-Cell Isolation from Adipose Tissue
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Mice were euthanized by isoflurane overdose and cervical dislocation and perfused with 20 ml PBS through the left ventricle. eAT pads were collected, minced, and digested in 6 ml of 2-mg/mL type II collagenase (Worthington # LS004177) for 30 min at 37 °C. Digested eAT was then vortexed, filtered through 100 μm filters, lysed with ACK buffer, and filtered through 35 μm filters as previously described81 .
The AT stromal vascular fraction was prepped with anti-mouse Fc Block (BD Biosciences) at 1:200. Cells from each mouse were labeled with unique hashtag antibodies (1:200) (Biolegend TotalSeq-C) and anti-CD45 microbeads (10 μL/ sample) (Miltenyi # 130-052-301). Biological replicates were pooled and sorted on a Miltenyi AutoMACs using the “possel_s” option. CD45+ cells were then labeled for CITE-sequencing using TotalSeq-C antibodies (Biolegend) for cell surface markers to identify major cell types. All immunolabeling was completed at a 1:200 dilution for 20 min at 4 °C in the dark. More information regarding specific samples and antibody manufacturer/catalog numbers can be found in Supplementary Table1 . Cells were stained with 0.25 µg/mL DAPI for FACS sorting and DAPI− viable cells were collected for downstream processing and sequencing.
The AT stromal vascular fraction was prepped with anti-mouse Fc Block (BD Biosciences) at 1:200. Cells from each mouse were labeled with unique hashtag antibodies (1:200) (Biolegend TotalSeq-C) and anti-CD45 microbeads (10 μL/ sample) (Miltenyi # 130-052-301). Biological replicates were pooled and sorted on a Miltenyi AutoMACs using the “possel_s” option. CD45+ cells were then labeled for CITE-sequencing using TotalSeq-C antibodies (Biolegend) for cell surface markers to identify major cell types. All immunolabeling was completed at a 1:200 dilution for 20 min at 4 °C in the dark. More information regarding specific samples and antibody manufacturer/catalog numbers can be found in Supplementary Table
3
Isolation and Characterization of Mouse Skeletal Stem Cells
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Mice were sacrificed at a time indicated in each experiment. Bones spanning fracture sites were collected, muscles were removed, and bone marrows were flushed with PBS. These collected bones spanning fracture sites were designated as fractured bones. In addition, the contralateral bones without muscles and the bone marrows were included as controls and designated as intact bones. Fractured bones and intact bones (about 3 mm) were cut into small pieces and digested with collagenase (2 mg/ml) for 1 hour at 37° C, and MNCs were collected. The MNCs were either further purified for CD90+ mSSCs or directly used for FACS analyses.
To purify CD90+ mSSCs, CD45+ cells were first removed using anti-CD45 Microbeads (Miltenyi Biotec). Then, CD90+ cells in the CD45− cells were positively purified using Dynabeads Mouse Pan T (Thy1.2) (Thermo Fisher Scientific), followed by removal of the beads. Our data showed that the bead-free CD45−CD90+ cells in the fractured bones were mostly AlphaV+. Hence, these purified cells were considered to be CD90+ mSSCs, i.e., CD45−Tie2−AlphaV+CD90+ cells.
To purify CD90+ mSSCs, CD45+ cells were first removed using anti-CD45 Microbeads (Miltenyi Biotec). Then, CD90+ cells in the CD45− cells were positively purified using Dynabeads Mouse Pan T (Thy1.2) (Thermo Fisher Scientific), followed by removal of the beads. Our data showed that the bead-free CD45−CD90+ cells in the fractured bones were mostly AlphaV+. Hence, these purified cells were considered to be CD90+ mSSCs, i.e., CD45−Tie2−AlphaV+CD90+ cells.
4
Isolation of Schwann Cells from Mouse Adipose
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From N=5 BTBR ob/ob and N=5 BTBR wild type mice, bilateral inguinal subcutaneous white adipose depots were pooled for each animal. Tissues were placed in an enzyme digest containing 2mg/mL collagenase A (Sigma-Aldrich, Cat#10103586001), 2mg/mL dispase II (Sigmam Cat#D4693) , and 0.05% Trypsin (Gibco, Cat#25200056). Tissues were dissociated for 30 minutes at 37°C using a gentleMACS Octo Dissociator with Heaters (Miltenyi, Cat#130096427). Cells were passed through a 100μm filter and spun down to pellet stromal vascular fraction (SVF). Cells were resuspended in buffer containing anti-CD45 microbeads (Miltenyi, Cat#130052301) and incubated for 15 minutes at 4°C. Cells were washed and incubated for 10 minutes at room temperature with anti-Pref1 antibody (Cell Signaling, Cat#2069) followed by incubation with anti-Rabbit IgG microbeads (Miltenyi, Cat#130048602) for 15 minutes at 4°C. Cells were passed through Large cell columns (Miltenyi, Cat# 130042202) in a magnetic field and unlabeled flow-through (Cd45-, Pref-1-) was collected. Collected cells were incubated with anti-O4 microbeads (Miltenyi, Cat#130096670). Cells were passed through a Large cell column (Miltenyi, Cat# 130042202) in a magnetic field and O4+ Schwann cells were collected from column in 300 μl of Trizol.
5
Magnetic Sorting for Macrophage and Fibroblast Isolation
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After IWAT and GWAT SVF isolation, cells were further separated into different subsets with magnetic activated cell sorting on OctoMACS™ separator (Miltenyi Biotec, Germany) per manufacturer’s instructions. Briefly, cells were incubated with anti-F4/80 MicroBeads UltraPure (Miltenyi Biotec, Germany) for 15 min in dark and on ice. After two washes in MS buffer (PBS, 0.5% BSA, 2mM EDTA), cells were loaded onto pre-rinsed MS columns (Miltenyi Biotec, Germany) placed in the magnetic field. Columns were rinsed two more times with MS buffer, and entire flow-through containing unlabeled cells was collected. Then, column was removed from magnetic separator and placed on collection tube. After adding MS buffer, magnetically labelled cells were flushed out with plunger. For fibroblast separation, F4/80- cells were first depleted from leukocytes with anti-CD45 MicroBeads (Miltenyi Biotec, Germany). Negative fraction was subsequently incubated with anti-CD90.2 MicroBeads (Miltenyi Biotec, Germany). Cells were resuspended in Qiazol (Qiagen, Germany) for RNA isolation. Purity of all sorted cells (macrophages, F4/80+), fibroblasts (F4/80-CD45-CD90.2+) and all negative cells was confirmed with quantitative real-time PCR.
6
Transcriptional Profiling of Tumor-T Cell Cocultures
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Cocultures of T lymphocytes and infected tumor cells were collected after 5 days. Tumor cells and T lymphocytes were then isolated via depletion strategy using anti-CD45 MicroBeads (Miltenyi, Bergisch Gladbach, Germany) to obtain tumor cells and anti-B7H3 PE (BD Bioscience), followed by anti-PE MicroBeads (Miltenyi) staining for the T-cell population. RNA was isolated using RNeasy Mini Kit (QIAGEN) and reversely transcribed with the cDNA SuperScript VILO cDNA Synthesis Kit (Life Technologies). Gene expression was then tested using TaqMan OpenArray Pathways Panels (Life Technologies) using 12K qPCR QuantStudio Flex Real-Time PCR system. Tumor cells were analyzed with the ‘human cancer panel’, while T cells were analyzed with ‘human signal transduction and inflammation panels’.
7
Isolation of Bone-Derived Stromal Cells
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For preparation of BLCs, femurs and tibias were harvested, cleaned of excess muscle tissue, and placed into PBS/5% FCS. Bones were crushed in a mortar, and BM was removed for separate processing of central BM. The bones were fragmented with scissors and transferred to a 15-ml tube (Falcon). Bones were spun down, and excess PBS/FCS was removed. Cells were isolated by incubation at 37°C with type I collagenase (3 mg/ml; Worthington Biochemical Corporation) for 45 min with rotation in DMEM with 10% FCS. The cell suspension was collected by passing through a 70-µm cell strainer. Collagenase was then added for a further 45 min. Cells were collected and washed in PBS/5% FCS/2 mM EDTA to reduce clumping.
For analysis of central BM stromal cells, BM cells were incubated at 37°C with type IV collagenase (2 mg/ml; Worthington Biochemical Corporation) for 20 min with rotation in DMEM with 10% FCS, and RBCs were lysed with ammonium chloride solution (STEMCELL Technologies) at room temperature for 10 min. Subsequently, CD45+ cells were depleted with anti-CD45 MicroBeads (Miltenyi Biotec). Cells were collected and resuspended in PBS/5% FCS/2 mM EDTA for FACS analysis.
For analysis of central BM stromal cells, BM cells were incubated at 37°C with type IV collagenase (2 mg/ml; Worthington Biochemical Corporation) for 20 min with rotation in DMEM with 10% FCS, and RBCs were lysed with ammonium chloride solution (STEMCELL Technologies) at room temperature for 10 min. Subsequently, CD45+ cells were depleted with anti-CD45 MicroBeads (Miltenyi Biotec). Cells were collected and resuspended in PBS/5% FCS/2 mM EDTA for FACS analysis.
8
Isolation and Sequencing of Lymphoid Stromal Cells
9
Murine Thymic Epithelial Cell Isolation
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Murine TEC isolation was performed at the indicated time points following it-LV treatment. Murine thymus was cleaned of fat and stromal tissue, and then digested at 37°C with an enzymatic solution containing Liberase TL and DNAse I. Digested tissues were collected in DMEM with 10% fetal bovine serum, 1% glutamine, and 1% penicillin and streptomycin. Single thymic cell suspensions were then incubated with anti-CD45 micro-beads (Miltenyi Biotec) and processed with the AutoMACS Pro Separator (Miltenyi Biotec). The CD45 negative fraction was retrieved and then tested by multiparametric cytofluorimetric analysis for the expression of TEC markers.44 (link)
To check TEC enrichment after isolation, cells were immune-stained by anti-CD45 (clone 30-F11, BioLegend), anti-EpCAM (clone G8.8, BioLegend) and pan anti-major MHC class II (IA/IE MHC-II) (clone M5/114.15.2), anti-CD49F (GoH3) (BioLegend), anti-Ly5.1 (clone 6C3) (Miltenyi), and anti-CD80 (16-10A1) (BD Pharmingen). Exclusion of dead cells was done by adding 200 μg/mL of DAPI solution just before acquisition.
To check TEC enrichment after isolation, cells were immune-stained by anti-CD45 (clone 30-F11, BioLegend), anti-EpCAM (clone G8.8, BioLegend) and pan anti-major MHC class II (IA/IE MHC-II) (clone M5/114.15.2), anti-CD49F (GoH3) (BioLegend), anti-Ly5.1 (clone 6C3) (Miltenyi), and anti-CD80 (16-10A1) (BD Pharmingen). Exclusion of dead cells was done by adding 200 μg/mL of DAPI solution just before acquisition.
10
Endometrial Cell Isolation and Culture
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Endometrial single-cell isolation was carried out as described [9 (link)]. Endometrial tissues were collected and the following procedure was performed with 24 h of the collection. Tissues were minced into pieces smaller than 1 mm and suspended in PBS containing collagenase III (0.3 mg/mL, Worthington, Lakewood, NJ, USA) and deoxyribonuclease I (40 ug/mL, SolarBio, Beijing, China). Tissue suspension was digested at 37 °C in water bath under constant shaking. Red blood cells were removed by density-gradient centrifugation using Ficoll-Paque (GE Healthcare, Berlin, Germany). Leukocytes were excluded using anti-CD45 microbeads (Miltenyi Biotec Inc., Bergisch Gladbach, Germany). Negative cells were subjected to subsequent isolation for epithelial and stromal cells. Epithelial cells were selected using anti-CD326 microbeads (Miltenyi). Negative cells were considered as stromal cells. Stromal cells were cultured in fibronectin-coated plates (1 mg/mL, Invitrogen). Culture media contained DMEM/F12, 10% FBS, 1% antibiotics, and 2 mmol/L L-glutamine (Invitrogen, Carlsbad, CA, USA). Cell culture was incubated in a humidified carbon dioxide (5%) incubator at 37 °C.
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