Human cd45 microbeads

Manufactured by Miltenyi Biotec

Sourced in Germany

Human CD45 MicroBeads are magnetic beads coated with antibodies specific to the human CD45 antigen. They are designed for the isolation or depletion of CD45-positive cells from various biological samples.

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Lab products found in correlation

Ls column, by Miltenyi Biotec (2 mentions) Clone 9c4, by BioLegend (1 mentions) Y 27632, by Bio-Techne (1 mentions) Facsaria 2, by BD (1 mentions) Cd45 microbeads, by Miltenyi Biotec (1 mentions) Human cd8 microbeads, by Miltenyi Biotec (1 mentions) Human cd4 microbeads, by Miltenyi Biotec (1 mentions) Interleukin 7 (il 7), by Thermo Fisher Scientific (1 mentions) Cd8 microbead, by Miltenyi Biotec (1 mentions) Cell strainer, by BD (1 mentions) Gentlemacs dissociator, by Miltenyi Biotec (1 mentions) Tumor dissociation kit, by Miltenyi Biotec (1 mentions) Pe mouse anti human cd61, by BD (1 mentions) Anti bcl 2, by Cell Signaling Technology (1 mentions) Prostaglandin e1 pge1, by Merck Group (1 mentions) Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (1 mentions) Taqman universal master mix 2, by Thermo Fisher Scientific (1 mentions) Adenosine diphosphate (adp), by Helena Laboratories (1 mentions) Rabbit anti β actin antibody, by Cell Signaling Technology (1 mentions) Mirvana mirna isolation kit, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

Human CD45 MicroBeads reagent Anti-human CD45 microbeads CD45 Microbeads non-human primate Anti-human CD45–conjugated microbeads CD45 MicroBeads

8 protocols using human cd45 microbeads

Immune Cell Depletion and Sorting

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For fetal and postnatal tissues, single cells from digested tissue were resuspended in MACS buffer containing 10 μM of the ROCK inhibitor Y-27632 (Tocris). Human CD45 MicroBeads (Miltenyi) were used to deplete immune cells according to the manufacturer’s instructions with the following modification: 5 μL of CD45 MicroBeads per 10⁷ total cells were added instead of 20 μL. LD columns were used for depletion and the CD45-negative fraction was collected in MACS buffer. Stromal cells from adult thymus were enriched using fluorescence-activated cell sorting (FACS). Blocking was done with human Fc receptor binding inhibitor monoclonal antibody (eBioscience) followed by staining for 20 min using human-specific antibodies against EPCAM (Clone 9C4, BioLegend) and CD45 (Clone HI30, Biolegend). After staining, cells were washed and resuspended in FACS buffer containing DAPI. Cells were sorted on BD FACS Aria II. Pre-gating was first done for live cells based the DAPI stain. The percentages of EPCAM⁺, EPCAM⁻CD45⁻, and CD45⁺ cells in each sample was determined by flow cytometric analysis or by calculating the ratio of cells in epithelial clusters, immune cluster, or other clusters over the total number of cells in the single-cell RNA-seq dataset.

Bautista J.L., Cramer N.T., Miller C.N., Chavez J., Berrios D.I., Byrnes L.E., Germino J., Ntranos V., Sneddon J.B., Burt T.D., Gardner J.M., Ye C.J., Anderson M.S, & Parent A.V. (2021). Single-cell transcriptional profiling of human thymic stroma uncovers novel cellular heterogeneity in the thymic medulla. Nature Communications, 12, 1096.

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Enrichment of Immune Cell Subsets

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Single-cell suspensions were thawed for each patient and filtered through a 40-μm filter into complete media (described above). Samples were enriched for CD8⁺, CD4⁺, and CD45⁻ cells (on ice) using three sequential rounds of positive selection by magnetic bead separation using MicroBeads (Miltenyi) according to the manufacturer’s protocol. Briefly, cells were resuspended in cell enrichment buffer (described above) and counted. Cells were incubated at 4°C for 15 min with Human CD8 MicroBeads, Human CD4 MicroBeads, or Human CD45 MicroBeads (Miltenyi) and then washed with cell enrichment buffer. Samples were passed through the LS column (Miltenyi), and both the positive and negative fractions were collected. To reduce the duration and maximize the cell recovery steps, CD8⁻ fraction was subsequently used for a second round CD4⁺ enrichment, and the CD4⁻ fraction was used for the subsequent CD45⁻ enrichment. Solutions were kept on ice throughout the duration of the separation.

Kilgour M.K., MacPherson S., Zacharias L.G., Ellis A.E., Sheldon R.D., Liu E.Y., Keyes S., Pauly B., Carleton G., Allard B., Smazynski J., Williams K.S., Watson P.H., Stagg J., Nelson B.H., DeBerardinis R.J., Jones R.G., Hamilton P.T, & Lum J.J. (2021). 1-Methylnicotinamide is an immune regulatory metabolite in human ovarian cancer. Science Advances, 7(4), eabe1174.

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Metabolomic profiling of expanded CD8+ T cells

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PBMCs were isolated from a leukapheresis product (STEMCELL Technologies) using Ficoll gradient density centrifugation. CD8⁺ T cells were isolated from the PBMCs using CD8 MicroBeads (Miltenyi) and expanded using TransAct (Miltenyi) for 2 weeks in complete media according to the manufacturer’s instructions. Cells were rested for 5 days in complete media with IL-7 (10 ng/ml; PeproTech) and then restimulated with TransAct. On day 7, cells were enriched using Human CD45 MicroBeads (Miltenyi) in three rounds of sequential enrichment according to the manufacturer’s instructions. Cells were aliquoted for analysis by flow cytometry (described above), and 1 million cells were aliquoted in triplicate for analysis by LC-MS/MS. Samples were processed for LC-MS/MS as described below. We imputed missing metabolite values with an ion count of 1000. Each sample was sum normalized by total ion count (TIC), log transformed, and auto-normalized in MetaboAnalystR before analysis.

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In Vivo Glucose/Fructose Tracing in Leukemia

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For the in vivo tracing experiments, 8–12 weeks old females NOD.Cg-Prkdc^scidIl2rg^tm1Wjl/SzJ mice (NSG, The Jackson Laboratory) were transplanted intravenously with 1×10⁶ MOLM13 cells. At clear signs of diseases (hunchback, weight loss, appearance of extra-medullary masses) the mice were injected intraperitoneally (IP) with 4 g/kg of [2-¹³C] glucose or fructose dissolved in PBS and sacrificed 5 hours later. The group of mice treated with NCT-503 was injected IP with the drug at 40 mg/kg 30 minutes prior to the sugar injection. A second drug treatment was administered after 3 hours from the sugar injection in order to match the shelf-life of NCT503 and keep the enzyme inhibited. At sacrifice, bone marrow was harvested and processed by crashing the bones in cold PBS. Human cells were positively selected using human CD45 MicroBeads (Miltenyi Biotec) following the manufacturer instruction. Auto MACS pro separator was used for the procedure. After separation, positive enriched human CD45 were counted and a dry pellet was snap frozen and stored at −80°. The same procedure was applied to mice engrafted with Patient 5-derived leukemic cells, the day of sacrifice. Mice engrafted with Patient 9-derived leukemic cells were subjected to the same treatment except the human engraftment was over 95% and enrichment with CD45 beads was not needed (experimental workflow is shown in Figure S7F).

Jeong S., Savino A.M., Chirayil R., Barin E., Cheng Y., Park S.M., Schurer A., Mullarky E., Cantley L.C., Kharas M.G, & Keshari K.R. (2020). High fructose drives the serine synthesis pathway in acute myeloid leukemic cells. Cell metabolism, 33(1), 145-159.e6.

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Isolation of CD45+ Cells from Liver Metastases

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Tumour specimens (liver metastases) from 10 patients (Table S1) were processed as previously described [9 (link)] with the GentleMACS Dissociator (Miltenyi Biotec, Bologna, Italy, cat# 130-093-235) after being treated with the Tumor Dissociation Kit (Miltenyi Biotech, cat# 130-095-929). After this procedure, the tumour cells were filtered (Cell Strainers, Falcon, Corning Stone, Staffordshire, England, cat# 352350) to remove clusters and to obtain a single-cell suspension before magnetic separation. The cells were then checked for viability by trypan blue dye exclusion and resuspended in buffer containing phosphate-buffered saline (PBS, Euroclone, Pero, Italy, cat# ECB4004L) pH 7.2, 0.5% bovine serum albumin (BSA, Kedrion Biopharma, Barga, Italy) for the isolation of CD45-positive cells, using human CD45 microbeads (cat# 130-118-780) and an LS Column (cat# 130-042-401) (Miltenyi Biotec), according to the manufacturers’ instructions. Negative and positive cells were collected, centrifuged and checked for viability and recovery.

Faris P., Rumolo A., Tapella L., Tanzi M., Metallo A., Conca F., Negri S., Lefkimmiatis K., Pedrazzoli P., Lim D., Montagna D, & Moccia F. (2022). Store-Operated Ca2+ Entry Is Up-Regulated in Tumour-Infiltrating Lymphocytes from Metastatic Colorectal Cancer Patients. Cancers, 14(14), 3312.

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Analyzing Platelet Apoptosis Mechanisms

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Human CD45 microbeads were obtained from Miltenyi Biotec (Bergisch Gladbach, Germany). Prostaglandin E₁ (PGE₁), and JC-1 were purchased from Sigma-Aldrich (St. Louis, MO, USA). FITC Annexin V Apoptosis Detection Kit and PE Mouse Anti-Human CD61 were obtained from BD Biosciences (Heidelberg, Germany). VerifyNow P2Y12 assay was purchased from Accumetrics (San Diego, CA, USA), and adenosine diphosphate (ADP) was from Helena Laboratories (Beaumont, TX, USA). Anti-Bcl-2, anti-Caspase-3, and anti-β-actin rabbit antibody were sourced from Cell Signaling Technology (Danvers, MA, USA). ABT-737 was from Selleck Chemical (Houston, TX, USA). mirVana miRNA Isolation Kit, Taqman microRNA primers (hsa-miR-145-5p and hsa-miR-15b-5p), Taqman microRNA Reverse Transcription Kit, Taqman Universal Master Mix II, mirVana miRNA mimic and inhibitor (hsa-miR-15b-5p), mirVana miRNA mimic and inhibitor negative control #1, and Lipofectamine RNAiMAX reagents were purchased from Applied Biosystems (Foster City, CA, USA).

Wang J., Yao Y., Zhang J., Tang X., Meng X., Wang M., Song L, & Yuan J. (2020). Platelet microRNA-15b protects against high platelet reactivity in patients undergoing percutaneous coronary intervention through Bcl-2-mediated platelet apoptosis. Annals of Translational Medicine, 8(6), 364.

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Isolating CD45-Negative Cells from Pancreatic Cancer Tumor Tissue

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We isolated CD45-negative cells from the single-cell suspension obtained from fresh pancreatic cancer tumor tissues using magnetic-activated cell sorting (MACS; Miltenyi Biotec). After thawing the cryopreserved single-cell suspensions, we depleted cell debris, dead cells, and dying cells using the Dead Cell Removal Kit (Miltenyi Biotec). Next, we labeled CD45-positive cells in live single-cell suspensions using human CD45 MicroBeads (Miltenyi Biotec) and sorted the unlabeled CD45-negative cells by MACS. Only the flowthroughs proceeded to single-cell library construction. For three of the PDAC patient surgical samples, CD45-positive cells were also collected and processed with CD45-negative cells.

Kim S., Leem G., Choi J., Koh Y., Lee S., Nam S.H., Kim J.S., Park C.H., Hwang H.K., Min K.I., Jo J.H., Lee H.S., Chung M.J., Park J.Y., Park S.W., Song S.Y., Shin E.C., Kang C.M., Bang S, & Park J.E. (2024). Integrative analysis of spatial and single-cell transcriptome data from human pancreatic cancer reveals an intermediate cancer cell population associated with poor prognosis. Genome Medicine, 16, 20.

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Leukemic cell-MSC adhesion and migration

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For adhesion, MSCs were plated in six-well plates and cultured for 24 h. Then, leukemic cells were cultured on the surface of MSCs for another 48 h with or without PW21. Attached leukemic cells were counted by flow cytometry after staining with human CD45 MicroBeads (Miltenyi Biotec, Germany) in the presence of CountBright absolute counting beads (Thermo Fisher Scientific, Waltham, MA, USA). Adhesion capability was defined according to the manufacturer’s instructions.
For migration, MSCs were plated in the lower chamber of transwell plates and cultured for 24 h. Leukemic cells were added in the insert and cultured for another 12 h with or without PW21. The cell number of migration was defined as viable cells in the lower chamber.

Wang J.D., Zhang W., Zhang J.W., Zhang L., Wang L.X., Zhou H.S., Long L., Lu G., Liu Q, & Long Z.J. (2020). A Novel Aurora Kinase Inhibitor Attenuates Leukemic Cell Proliferation Induced by Mesenchymal Stem Cells. Molecular Therapy Oncolytics, 18, 491-503.

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