For western blotting, CD4+ T cells were positively enriched using the EasySep cell isolation kit (Stemcell Technologies). In some experiments, enriched cells were cultured in RPMI-1640 medium containing 2% FBS with or without 20% B16 TCM at 37 °C. Cells were lysed in 1% NP-40 lysis buffer containing protease inhibitor cocktail (Sigma-Aldrich) and 1 mM sodium orthovanadate (Sigma-Aldrich). Protein lysates (5 – 20 µg) were subjected to SDS-PAGE, probed with indicated antibodies, and detected using an enhanced chemiluminescence substrate (Pierce). Polyclonal antibodies against STAT3 and S1PR1 were purchased from Santa Cruz Biotechnology. Monoclonal β-Actin antibody was purchased from Sigma-Aldrich. Phospho-STAT3 (Y705) antibody was purchased from Cell Signaling Technology.
Easysep cell isolation kit
Manufactured by STEMCELL
Sourced in Canada
The EasySep cell isolation kit is a product designed for the rapid and efficient isolation of specific cell types from heterogeneous samples. The kit utilizes a magnetic separation technology to selectively label and isolate the desired cell population, allowing for further downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Lymphoprep, by STEMCELL (2 mentions)
β actin monoclonal antibody, by Merck Group (1 mentions)
Phospho stat3 y705 antibody, by Cell Signaling Technology (1 mentions)
Polyclonal antibodies against, by Santa Cruz Biotechnology (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Sodium orthovanadate, by Merck Group (1 mentions)
Globalfiler pcr amplification kit, by Thermo Fisher Scientific (1 mentions)
Veriti 96 well thermal cycler, by Thermo Fisher Scientific (1 mentions)
Abi 3500xl genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Rosettesep b lymphocyte enrichment kit, by STEMCELL (1 mentions)
Rpmi 1640 medium, by Euroclone (1 mentions)
Dmem medium, by Thermo Fisher Scientific (1 mentions)
Hs5 human bone marrow stromal cell line, by American Type Culture Collection (1 mentions)
Cryostor cs10, by STEMCELL (1 mentions)
Sepmate 50, by STEMCELL (1 mentions)
Anti cd14 microbeads, by Miltenyi Biotec (1 mentions)
6 protocols using easysep cell isolation kit
1
Western Blotting of STAT3 and S1PR1
2
DNA Chimerism Assessment Protocol
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Assessment of DNA chimerism was performed using the GlobalFiler™ PCR Amplification Kit (Applied Biosystems, Cat#4476135, Waltham, MA, USA). The GlobalFiler™ kit utilizes 21 autosomal STR markers (D3S1358, vWA, D16S539, CSF1PO, TPOX, D8S1179, D21S11, D18S51, D2S441, D19S433, TH01, FGA, D22S1045, D5S818, D13S317, D7S820, SE33, D10S1248, D1S1656, D12S391, D2S1338) plus Amelogenin as a sex determining marker with DYS391 and Y-indel as Y-chromosome markers. For each STR marker, separate PCR reactions were carried out with both positive and negative controls. PCR amplification was performed on the VERITI, 96 well thermal cycler (Applied Biosystems, USA) following the manufacturer’s recommended protocols. Assessment of the STR fragments was performed on the ABI 3500XL Genetic Analyzer (Applied Biosystems, USA) following the manufacturer’s instructions. Final chimerism determinations were performed using ChimeRMarker™ Genetic Analysis software (SoftGenetics, State College, PA. USA). CD3 and CD33 subsets were isolated from whole blood via magnetic beads using the EasySep™ cell isolation kit (Cat#18981RF and #17885RF) and the RoboSep™ automated cell separator system (StemCell Technologies, Cambridge, MA.). Cell purity was assessed by flow cytometry and was >95%.
3
Naive and Effector T Cell Isolation
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Human naïve CD4+ T cells, effector T (Teff) cells and dendritic cells (DCs) were further selected from PBMCs using the EasySep cell isolation kit (StemCell Technologies, Vancouver, British Columbia, Canada). Naïve CD4+ T and Teff cells were defined as the CD4+CD45RA+ T cells and CD4+CD25− T cells, respectively. The cell purity was greater than 96%.
4
Isolation and culture of primary B cells
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The HS5 human bone marrow stromal cell line was obtained from the American Type Culture Collection (ATTC) and cultured in DMEM medium (GIBCO) supplemented with 10% v/v FBS, 15 mg/mL Pen/Strep at 37°C and 5% CO2. Primary leukaemic, from patients, and healthy, from donors, CD19+ B cells were negatively selected from fresh PB and BM using the RosetteSep B lymphocyte enrichment kit (Stemcell Technologies) and separated via density gradient Lymphoprep (Stemcell Technologies). Healthy B cells were then isolated with EasySep Cell isolation kit (Stemcell Technologies). B cells were isolated from lymphoid tissues after mechanical smashing to recover the cells in suspension and later purified following the same protocol described for healthy B cells. The purity of all the isolated B cells was >98%. Primary cells were cultured in RPMI 1640 medium (EuroClone) supplemented with 10% (v/v) Fetal Bovine Serum (FBS) and 15 mg/mL Pen/Strep (complete RPMI) at 37°C and 5% CO2.
5
Adoptive T cell transfer to obese mice
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Spleen CD4+ and CD8+ T cells were purified using EasySep cell isolation kits (StemCell Technologies). Obese Rag1 KO BMCs received two weekly intravenous injections of 5 × 106 CD4+ T cells from WT and 5 × 106 CD8+ T cells from either WT or March1 KO mice.
6
Isolation and Cryopreservation of Immune Cells
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Leukocyte concentrates of healthy donors were purchased from the local blood bank at Skåne university hospital, department of Clinical Immunology and Transfusion Medicine, Lund, Sweden with the approval of the Swedish Ethical Review Authority (Etikprövningsmyndigheten). Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Lymphoprep™ and SepMate™-50 (STEMCELL Technologies, Vancouver, Canada). CD14+ monocytes were magnetically enriched from the isolated PBMCs using anti-CD14 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). CD4+ T cells, B cells or total T cells (CD4+ and CD8+) were isolated from PBMCs via immunomagnetic negative selection using EasySep™ Cell Isolation Kits (STEMCELL Technologies). All cells were isolated according to manufacturer’s instructions. Isolated PBMCs, PBMCs depleted of CD14+ cells, and generated DCs were separately cryopreserved in CryoStor® CS10 (STEMCELL Technologies).
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