Human peripheral blood mononuclear cells (PBMCs) were collected in compliance with the protocols outlined by the Washington University School of Medicine Human Studies Committee and harvested by ficoll (GE Healthcare Bio-science AB, Uppsala, Sweden) gradient centrifugation. Human pan T cells were isolated from the human PBMCs using Miltenyi microbeads and AutoMACS (Miltenyi Biotech, Auburn, CA) (10 (link)). Mouse pan T cells were isolated from mouse spleens using Miltenyi microbeads and an AutoMACS (Miltenyi Biotech) (10 (link)).
Miltenyi microbeads
Manufactured by Miltenyi Biotec
Miltenyi MicroBeads are small, uniform, and superparamagnetic particles designed for cell separation and purification applications. They are used in conjunction with magnetic separation technology to isolate specific cell populations from complex biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Automacs, by Miltenyi Biotec (1 mentions)
Ficoll, by GE Healthcare (1 mentions)
Midimacs separation unit, by Miltenyi Biotec (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (1 mentions)
Trypan blue, by Merck Group (1 mentions)
4 protocols using miltenyi microbeads
1
Isolation of Human and Mouse T Cells
2
Thymic Stromal Cell Isolation
3
Nanoparticle Biodistribution in Tumor Mice
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Two weeks after tumor cell inoculation, the Huh7 tumor-bearing mice were used for nanoparticle distribution analysis. For the chloroquine pretreated group, chloroquine phosphate at a dose of 40 mg/kg/day was intraperitoneally injected for 3 continuous days. In vivo distribution studies were performed with i.v. injection of Rhodamine B-labeled nanoparticles (10 mg/mL, n = 5). Mice were anesthetized for abdominal incision. Then, the liver was perfused and parenchyma cells and non-parenchyma cells fractions were separated through centrifugation at 50×g for 5 min at 4 °C. Cells were resuspended with MACS buffer and mixed with each Miltenyi MicroBeads (#130-110-187 or #130-110-443, Miltenyi) and mixed gently. Parenchyma cells and non-parenchyma cells were proceeded to magnetic separation according to the manual. Therefore, parenchyma cells were divided into tumor cells and other parenchymal cells. Non-parenchyma cells were divided into Kupffer cells and other non-parenchymal cells. For flow cytometry, Rhodamine B positive events were detected with a 488 nm wavelength laser and a 585/42 optical filter. Gating strategy for nanoparticle positive events was set based on the nanoparticle untreated group.
4
Optimized Depletion of Immune Cells
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For comparative depletion experiments, heparinized blood was divided into aliquots and incubated with Miltenyi MicroBeads at 2–8°C. Volumes and incubation times were optimized for each antigen and achieved approximately 90% depletion of the target cell [anti-CD4: 100 mcl/ml, 30 min; anti-CD8: 50 mcl/ml, 15 min; anti-CD14: 100 mcl/ml, 30 min; anti-CD15: 50 mcl/ml, 15 min; Basic (unconjugated) MicroBeads used for controls: 50 mcl/ml, 15–30 min; see Figure S1 in Supplementary Material]. Blood was diluted 1:1 with RPMI-1640 and passed through Miltenyi Biotec LS columns supported in magnets (MidiMACS Separation Unit, Miltenyi Biotec); columns had been pre-“primed” with 3 ml MACS buffer [0.5% bovine serum albumin (Sigma) and 2 mM EDTA (Sigma)], all as previously described (2 (link)). Depleted blood was collected in Universal containers. Granulocyte isolation using discontinuous Percoll gradient was performed as previously described (2 (link)). Neutrophils were rendered necrotic (when required) via heat shock at 60°C for approximately 20 min, until all cells were trypan blue (Sigma) positive by microscopy, and then allowed to cool.
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