At 12–16 wk after transplant, hNSG-SGM3 mice were euthanized, and spleen and liver were collected for single-cell suspension. Spleen and liver were first digested with 25 µg/ml Liberase (Roche Diagnostics) for 10–30 min at 37°C; single-cell suspensions were made, and the debris was removed by filtering through a 70-µm cell strainer. Live cells were isolated using Ficoll-Paque Plus density gradient centrifugation. Human immune cells were enriched by using the Mouse/Human Chimera isolation kit (StemCell Technologies) following the manufacturer’s protocol. For the enrichment of CD33+ or CD33neg cells, enriched hCD45+ cells were further stained with PE-conjugated CD33 antibody (WM53; Biolegend) for 15 min and enriched by using EasySep PE selection kit (StemCell Technologies). For CD14, CD11b, CD66b, CD117, and FCER1A depletion, hCD45+ cells were further stained with PE-conjugated CD14 (MϕP-9; BD Biosciences), CD11b (IRCF44; Biolegend), CD66b (G10F5; Biolegend), CD117 (104D2; Biolegend), and FCER1A (AER-37; Biolegend) antibodies (Table S10 ) for 15 min and enriched by using the EasySep PE selection kit (StemCell Technologies). Isolated human immune cells had purity exceeding 95%. Human immune cells were adoptively transferred at 107 cells per mouse by tail-vein i.v. injection.
Easysep pe selection kit
Manufactured by STEMCELL
Sourced in Canada
The EasySep PE Selection Kit is a magnetic cell separation system designed to isolate target cells from a heterogeneous cell population. The kit utilizes magnetic particles coated with antibodies specific to the cell surface markers of interest, allowing for the positive selection of the desired cell type.
Automatically generated - may contain errors
Lab products found in correlation
Anti human cd45 pe antibody, by BD (3 mentions)
Dead cell removal kit, by Miltenyi Biotec (2 mentions)
Collagenase 4, by Merck Group (2 mentions)
Dnase 1, by Roche (2 mentions)
Trypsin, by Thermo Fisher Scientific (2 mentions)
Liberase, by Roche (1 mentions)
Mouse human chimera isolation kit, by STEMCELL (1 mentions)
Ssoadvanced universal sybr green supermix, by Bio-Rad (1 mentions)
Complete freund s adjuvant, by Thermo Fisher Scientific (1 mentions)
Pertussis toxin, by List Biological Laboratories (1 mentions)
Percoll, by GE Healthcare (1 mentions)
Phosphate buffered saline (pbs), by Avantor (1 mentions)
Fetal bovine serum (fbs), by Avantor (1 mentions)
Mycobacterium tuberculosis strain h37ra, by BD (1 mentions)
L glutamine, by Avantor (1 mentions)
Hepes, by Avantor (1 mentions)
Rbc lysis buffer, by Merck Group (1 mentions)
Propidium iodide rnase staining solution, by Cell Signaling Technology (1 mentions)
Mirneasy mini kit, by Qiagen (1 mentions)
Rpmi 1640, by Avantor (1 mentions)
21 protocols using easysep pe selection kit
1
Isolation and Enrichment of Human Immune Cells from hNSG-SGM3 Mice
2
pDCs Isolation and Viral Infection
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DCs derived from CLPs using AC-6-feeder system for 16 d were stained with anti-CD11b-PE first, followed by negative selection for pDCs using EasySep PE-selection kit (Stemcell). Typically, the purity of pDCs after negative selection was between 92–95%. Purified pDCs at 1.2x105 were infected with or without VSV at an MOI of 10 for 18 h. The culture supernatant were collected and subjected to ELISA for IFN-α (PBL Assay Science) as described in the following.
3
Isolation and Functional Analysis of Tumor-Infiltrating Lymphocytes
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After digestion of harvested tumors, necrotic debris was removed from the cell suspension using a Dead Cell Removal Kit (Miltenyi Biotech, CA). TILs were subsequently isolated by positive selection using an anti-human CD45-PE antibody (BD Biosciences, CA) with the EasySEP PE Selection Kit (STEMCELL Technologies, Vancouver, Canada). Once isolated, functional analyses for TILs were performed in two different ways: (i) luciferase coculture killing assays, and (ii) measurement of antigen-induced T cell IFN-γ secretion by ELISA (see above). Pooling of samples was required in order to isolate sufficient numbers of viable TILs after all processing steps (i.e. harvest, digestion, single cell preparation via filtering and washing, dead cell removal, and CD45 magnetic separation) to perform ex-vivo co-culture killing experiments. When samples were pooled, at least three replicates were maintained within each group for statistics purposes.
4
Experimental Protocol for Mouse EAE Model
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The reagents used in this study were purchased as described: THC and CBD from Cayman Chemical (Michigan, USA), myelin oligodendrocyte glycoprotein (MOG35−55) peptide H-MEVGWYRSPFSRVVHLYRNGK-OH (PolyPeptide Laboratories, San Diego, CA, USA). Mycobacterium tuberculosis (strain H37Ra) (BD, Franklin Lakes, NJ, USA), complete Freund's adjuvant (Fisher, Hampton, NH, USA), Pertussis toxin (List Biological Laboratories, Campbell, CA, USA), Percoll, GE Healthcare Life Sciences (Pittsburgh, PA, USA); Neural Tissue Dissociation Kit (P) (Miltenyi Biotech, Auburn, CA, USA), RBC lysis buffer (Sigma-Aldrich, St. Louis, MO, USA), RPMI 1640, l-glutamine, HEPES, phosphate-buffered saline, and fetal bovine serum (VWR, West Chester, PA, USA), ELISA Max Kits IL-10, IL-17A, IFN-γ, IL-6, IL-1β, TNF-α, and TGF-β and FITC Annexin V/-PI apoptosis kit (Biolegend, San Diego, CA). EasySep PE selection kit (Stemcell Technologies, Cambridge, MA, USA), Propidium Iodide (PI)/RNase Staining Solution (Cell Signaling Technology, Danvers, MA, USA), miRNeasy Mini Kit, miScript II RT Kit and miRNAs primers (Qiagen, Valencia, CA), mRNAs primers (Integrated DNA technologies, Coralville, IA, USA) and SsoAdvanced™ Universal SYBR® Green Supermix (Bio-Rad, Hercules, CA, USA).
5
Isolation and Functional Analysis of TILs
6
Immunology Reagents and Cell Culture Materials
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Antibodies were from Tonbo Biosciences, Biolegend or BD Biosciences. 5-(and−6)-Carboxyfluorescein diacetate succinimidyl ester (CFSE) was from Tonbo Biosciences (San Diego, CA). EasySep PE selection kit was from STEMCELL (Vancouver, Canada). Purification columns HisTrap Crude FF and HiTrap Q FF were from GE Healthcare (Piscataway, NJ). U-PLEX kits were from Meso Scale Discovery (Rockville, MD). Double-color ELISPOT kits were from Cellular Technology Limited (Cleveland, OH). N,N-Dimethyldodecylamine N-oxide (LDAO) solution was from Sigma-Aldrich (St. Louis, MO). Limulus Amebocyte Lysate (LAL) endpoint chromogenic kit was from Lonza (Allendale, NJ). All cell culture plastics were TPP brand from MidSci. Recombinant premium-grade GM-CSF was from Miltenyi Biotec (San Diego, CA). RPMI-1640 and fetal bovine serum were from Mediatech (Manassas, VA).
7
Isolating TANs and TAMs from Tumor Tissues
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For TANs isolation, fresh ICC tissues were sliced into small pieces and digested in RPMI 1640 supplemented with 0.05% collagenase IV (Sigma-Aldrich), 0.002% DNase I (Roche) and 20% FBS at 37°C for 30 min. We filtered dissociated cells through a 150 µm mesh and then these cells were centrifuged at 2500 rpm for 20 min with 1 mL cell suspension and 10 mL Ficoll-Hypaque in a 15 mL tube. Thereafter, the leukocytes were harvested and CD66b+ (human neutrophils) were isolated using the EasySep PE Selection Kit (STEMCELL Technologies, Vancouver, Canada) according to the manufacturer’s protocol.
For TAMs isolation, tumor tissues were harvested and digested and single-cell suspensions were collected as mentioned before.22 (link) The leukocytes were harvested and CD14+ macrophages were isolated using CD14 MicroBeads (Miltenyi Biotec) according to the manufacturer’s protocol (online supplemental figure 2 ).
For TAMs isolation, tumor tissues were harvested and digested and single-cell suspensions were collected as mentioned before.22 (link) The leukocytes were harvested and CD14+ macrophages were isolated using CD14 MicroBeads (Miltenyi Biotec) according to the manufacturer’s protocol (
8
Depletion of Pericyte Fraction in SVF
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SVF cells freshly isolated as previously described underwent depletion of the pericyte components using the EasySep PE selection kit and an EasySep magnet (StemCell Technologies) according to the manufacturer’s protocol. Total SVF were depleted of CD146+ cells by positive selection following incubation with PE-CD146 antibody (Biolegend). The resulting SVF/CD146− cellular fraction was used for the experiments. Mock cells underwent the same sorting procedure but without the primary antibody and were used as control.
9
TIL Isolation and Functional Assays
10
Isolation and Transplantation of CD133+ Liver Progenitor Cells
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