Femurs and tibiae of Ubc-GFP mice were flushed with PBS + 2% FCS using a 21-gauge needle and a 10 mL syringe to collect the BM plug, which was then dispersed by reflux through the needle. The suspension was centrifuged 680 × g for 5 minutes at 4 °C. Cells were then lysed with lysis buffer (0.17M NH4Cl, 1 mM EDTA, 10 mM NaHCO3) at room temperature for 5 minutes, washed with 2 volumes PBS + 2%FCS, and centrifuged at 680 × g for 5 minutes. Cells were resuspended in PBS + 2%FCS and filtered through 40 μm nylon mesh. Cells were again centrifuged 680 × g for 5 minutes and treated with anti-CD16/32 antibody for Fc-receptor block (2 μL/mouse) for 5 minutes followed by addition of anti-CD45 magnetic beads (Miltenyi) at a 1/5 volume/volume ratio for 15 minutes. After removing the antibody with two PBS + 2%FCS washes, CD45-positive cells were isolated using Auto-MACS Pro (Miltenyi). Isolated cells were centrifuged once at 340 × g for 5 minutes. and injected into WT mice (3 × 106 cells/mouse).
Anti cd45 magnetic beads
Manufactured by Miltenyi Biotec
Sourced in Germany
Anti-CD45 magnetic beads are a laboratory product designed for cell separation and isolation. The beads are coated with antibodies specific to the CD45 cell surface antigen, which is expressed on most hematopoietic cells. These beads can be used to magnetically separate and enrich CD45-positive cells from complex samples, such as blood or tissue preparations.
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Lab products found in correlation
Dnase 1, by Roche (2 mentions)
Facsaria flow cytometer, by BD (2 mentions)
Sybr greener, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Quantitect reverse transcription kit, by Qiagen (2 mentions)
Automacs pro, by Miltenyi Biotec (1 mentions)
Anti epcam magnetic beads, by Miltenyi Biotec (1 mentions)
Cell separation columns, by Miltenyi Biotec (1 mentions)
Gfr matrigel, by BD (1 mentions)
Magnetic ls macs columns, by Miltenyi Biotec (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (1 mentions)
Ficoll paque plus, by GE Healthcare (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Hanks balanced salt solution (hbss), by Lonza (1 mentions)
Edta vacutainer, by BD (1 mentions)
Deoxyribonuclease 1, by Merck Group (1 mentions)
Propidium iodide, by Thermo Fisher Scientific (1 mentions)
Facsaria iiiu, by BD (1 mentions)
Facs sorp aria iiu, by BD (1 mentions)
Collagenase type 1, by Thermo Fisher Scientific (1 mentions)
17 protocols using anti cd45 magnetic beads
1
Isolating Bone Marrow Cells from Ubc-GFP Mice
2
Isolation of Murine Lung Cancer Cells
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KPV+/+ and KPV−/− mice were treated with Ad-Cre as described above; after 6 weeks, mice were sacrificed and lung tumors were excised. The tissue was dissociated into a single-cell suspension in 0.2 mg/mL DNase and 2 mg/mL collagenase D and filtered through a 40 μm filter. Cells then underwent two rounds of selection. First, cells were treated with anti-CD45 magnetic beads (Miltenyi Biotec, 130-052-301) and were passed through a magnetic column. CD45-negative cells were then subjected to anti-EpCAM magnetic beads (Miltenyi Biotec, 130-105-958) and underwent positive selection. CD45-negative, EpCAM-positive cells were expanded in vitro and were used in experiments between passages 1 and 10. Cells derived from a human lung adenocarcinoma (A549) were obtained from the American Type Culture Collection (ATCC, Manassas, VA). All cells were maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 µg/mL streptomycin, and HEPES buffer. All cells were grown in a humidified incubator of 5% CO2/95% air at 37 °C.
3
Marker-Independent CTC Enrichment Protocol
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1. Leucosep®Peripheral blood samples were collected in EDTA tubes. After performing plasma separation of the whole blood sample (described below) density gradient centrifugation with Leucosep™ tubes and Ficoll-Paque™ media was used to isolate the peripheral blood mononuclear cells (PBMCs) (800× g, 10 min). The mononuclear cell fraction was transferred to a new 50 mL tube, washed once and centrifuged for 15 min at 300× g, in order to form a cell pellet. After resuspending the cells in PBS, they were transferred to glass slides by cytospin centrifugation.
2. Negative selection/MACS(−)
Mononuclear cells were prepared as described above (positive selection). CD45 positive cells were depleted from the sample, using anti-CD45 magnetic beads, according to the manufacturer’s protocol (Miltenyi, Bergisch Gladbach, Germany).
3. Marker-independent CTC enrichment (ParsortixTM device)
Parsortix™ is a size and deformability-based method that allows for marker-independent CTC enrichment (Angle Plc, Guilford, UK). Cells were separated according to their size and deformability (final separation gap 8 µm), using a disposable cassette, according to our previous work [45 (link)].
2. Negative selection/MACS(−)
Mononuclear cells were prepared as described above (positive selection). CD45 positive cells were depleted from the sample, using anti-CD45 magnetic beads, according to the manufacturer’s protocol (Miltenyi, Bergisch Gladbach, Germany).
3. Marker-independent CTC enrichment (ParsortixTM device)
Parsortix™ is a size and deformability-based method that allows for marker-independent CTC enrichment (Angle Plc, Guilford, UK). Cells were separated according to their size and deformability (final separation gap 8 µm), using a disposable cassette, according to our previous work [45 (link)].
4
Adult Lung Single Cell Isolation
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We prepared adult lung single cell suspensions by enzymatic digestion and in some experiments depleted CD45+ cells by Cell Separation Columns (MACS) using for positive selection (LS) columns (Miltenyi Biotec) in MACS buffer (0.5% bovine serum albumin (BSA), 2 mM EDTA in sterile 1× PBS, filtered and degassed) according to the protocol provided by the vendor. CD45+ cells were depleted by treating cells by binding with anti‐CD45 magnetic beads (Miltenyi Biotec). Depleted cell populations were analyzed by FACS, plated on growth factor–reduced (GFR) Matrigel (BD) for colony‐forming assay as indicated in “Results” section.
5
Isolation of PBMC from NSCLC Patients
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Peripheral blood was collected from 19 patients with metastatic NSCLC (S1 Table ) with informed written consent under University of Wisconsin IRB approved protocol. A maximum of 50 mL of blood was obtained at any given blood draw using EDTA vacutainers (BD). Whole blood was diluted 1:1 with Hank’s balanced salt solution (HBSS, Lonza) and 30 mL of diluted blood was underlaid with 10 mL of ficoll-paque PLUS (GE Healthcare) per 50 mL conical tube. The blood was centrifuged for 40 min at 974 g, and resulting buffy coats were washed once with HBSS. Peripheral blood mononuclear cells (PBMCs) were incubated with commercially available, pre-conjugated, anti-CD45 magnetic beads (Miltenyi) in a buffer containing 2 mM EDTA (Fisher Scientific) and 0.5% bovine serum albumin (BSA, Sigma-Aldrich) in phosphate buffered saline (PBS, Hyclone), after which CD45+ cells were removed using magnetic LS MACS columns (Miltenyi).
6
Isolation and Characterization of Perivascular Mesenchymal Cells
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For analysis of MSCs, BM cells were isolated by flushing from femora and tibiae and were then enzymatically digested with 7 mg/mL collagenase type I (#17100-017; Invitrogen) and 1 mg/mL deoxyribonuclease I (D5025-750KU; Sigma-Aldrich) in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum (FCS) at 37°C for 20 min. Cells were collected and washed with PBS +2% FCS and centrifuged at 600 × g for 5 min at 4°C. The cells were resuspended in PBS +2% FCS and filtered through a 70-μm nylon mesh. The cells were then centrifuged again at 600 × g for 5 min and incubated with an anti-CD16/32 antibody (2 μL/mouse) for 5 min at 4°C for Fc receptor blockade. Anti-CD45 magnetic beads (Miltenyi) were then added at a 1/10 vol/vol ratio and incubated for 15 min at 4°C. After unbound antibody was removed by two washes with PBS +2% FCS, CD45+ cells were isolated using an AutoMACS Pro Separator (Miltenyi) with the Depletes-S program. The isolated cells were centrifuged at 340 × g for 5 min and stained with flow cytometry antibodies at 4°C. CD45−Ter119−CD31−LepR+CD140a+ cells were isolated as perivascular mesenchymal cells. Dead cells were eliminated by staining with 1 μg/mL propidium iodide (Invitrogen). Cell sorting was performed using an SORP FACS Aria IIu or FACS Aria IIIu instrument (BD Biosciences). Data were analyzed with FlowJo software (TreeStar).
7
Isolation and Culture of Mouse Bone Marrow Stromal Cells
8
Isolation and Purification of Immune Cells
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Thymocyte suspensions were prepared by manual disruption using glass slides. When mentioned, Foxp3pos cells were excluded based on GFP expression. BM were flushed from leg bone and depleted of CD3pos T cells using anti-CD3 magnetic beads (Miltenyi-Biotec). For mTEC isolation, thymic fragments were digested in PBS containing 1 mg/ml collagenase D (Sigma-Aldrich) and 2 μg/ml DNaseI (Roche). Remaining hematopoietic cells of the preparation were removed with anti-CD45 magnetic beads (Miltenyi Biotec). Both mTEC and thymocyte purification was performed by cell sorting on a FACSAria II (BD biosciences). For lamina propria, and skin, organs were was cut into small pieces and incubated with 5 mM EDTA, 1 mM DTT (Sigma-Aldrich) at 37 °C. Epithelial cells were separated from tissue. Tissues were then digested with 0.6 mg/ml of collagenase D (Sigma-Aldrich) and 100 µl of DNase I (Roche) and cells centrifugated on a 40% Percoll gradient (Fisher Scientific).
9
Isolation of Colonic Lamina Propria Cells
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Isolation of colonic LP cells was performed as previously described [18 (link)]. Briefly, colonic tissue was cut into pieces 0.5 cm in length and placed in an orbital shaker for 20 min at 37 °C in HBSS with 10% FCS and 2 mM EDTA. This shaking step was repeated and then the remaining tissue was minced and placed in HBSS with 10% FCS, 1 mg/mL collagenase IV (Sigma), and 40 U/mL DNase (Roche) I and shaken for 20 min at 37 °C. The contents are then filtrated through a 70 μm cell strainer directly into a 50 mL conical tube. Each 50 mL conical tube is topped off with HBSS/FBS and centrifuged at 4 °C. After the repetition of this step, the supernatant is poured off and the cell pellet is resuspended in ice-cold HBSS/FBS. From these resulting LP cells, CD45+ immune cells were isolated by using anti-CD45 magnetic beads (Miltenyi) and subsequently used for downstream analysis.
10
Isolation of Cardiac Cell Populations
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Different cell types were purified from the heart 2 days after LAD ligation. Cardiomyocytes were isolated as described56 (link). For endothelial cells, inflammatory cells and fibroblasts, hearts were digested to single cell suspension using the Skeletal Muscle dissociation kit (Miltenyi Biotech). Endothelial cells were positively selected using anti-CD31 coated magnetic beads (Miltenyi Biotech), following manufacturer instructions. CD31-depleted cells were subsequently incubated with anti-CD45 magnetic beads (Miltenyi Biotech) to separate inflammatory cells from cardiac fibroblasts. Purified cells were pelleted at 4 °C at 300 × g for 5 min for RNA extraction.
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