Jurkat T cells (TIB-152), HEK293T (CRL-1573), Raji B cells (CCL-86; all from ATCC), and Platinum-E (Plat-E) retroviral packaging cells (Cell Biolabs, San Diego, CA, USA) were maintained in RPMI-1640 or Dulbecco’s modified Eagle medium (Invitrogen) supplemented with 10% (v/v) fetal bovine serum (FBS; Invitrogen). Naive CD3+ T cells were purified from the mouse spleen and lymph nodes by negative selection using a T cell enrichment column (R&D Systems). To generate mouse T cell blasts, CD3+ T cells were incubated in 2 µg/mL anti-CD3/28-coated culture plates with 100 U/mL rIL-2 for 48 h and cultured for a further 5 days with 100 U/mL rIL-2. Mouse splenocytes were dispersed and purified into CD4+, CD8+, and CD19+ populations using the EasySep magnetic separation system (Stemcell Technologies, Vancouver, Canada) or MACS cell separation (Miltenyi Biotec, Bergisch Gladbach, Germany). The purity of each population was confirmed as >95% by flow cytometry. To establish B cell blasts, CD19+ cells from C57BL/6 wild-type mice were activated with LPS (10 μg/mL) for three days in complete RPMI. Bone marrow was flushed from femur and tibia bones, and bone-marrow-derived DCs (BMDCs) were grown with the addition of 20 ng/mL granulocyte macrophage-colony stimulating factor for five days.
Easysep magnetic separation system
Manufactured by STEMCELL
Sourced in Canada
The EasySep magnetic separation system is a laboratory instrument used for the isolation and enrichment of target cells from complex samples. It utilizes magnetic particles that bind to specific cell types, allowing for their separation from the rest of the sample using a magnetic field. The core function of this system is to enable efficient and reproducible cell isolation for various downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
T cell enrichment column, by R&D Systems (3 mentions)
Macs cell separation, by Miltenyi Biotec (2 mentions)
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Pcl neo mova, by Addgene (2 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (2 mentions)
Platinum e plat e retroviral packaging cells, by Cell Biolabs (1 mentions)
Hek293t, by American Type Culture Collection (1 mentions)
Platinum e, by Cell Biolabs (1 mentions)
B16f10 cells, by American Type Culture Collection (1 mentions)
Hek293t cell, by American Type Culture Collection (1 mentions)
Eo771 cells, by American Type Culture Collection (1 mentions)
Streptavidin coated magnetic beads, by Spherotech (1 mentions)
Phenylmethylsulfonyl fluoride pmsf, by Merck Group (1 mentions)
Aprotinin, by Merck Group (1 mentions)
Sodium orthovanadate, by Merck Group (1 mentions)
5 protocols using easysep magnetic separation system
1
Isolation and Cultivation of Diverse Immune Cells
2
Isolation and Activation of Immune Cell Populations
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Jurkat T (TIB-152), HEK293T (CRL-1573), and B16F10 (CRL-6475) cell lines were purchased from ATCC. Adult leukemia cell lines, MT2, and MT4 were purchased from CellBank Australia (Westmead, NSW, Australia). The retroviral ecotrophic packaging cell line Platinum-E was purchased from Cell Biolabs (San Diego, CA, USA). Cells were maintained in RPMI-1640 or Dulbecco’s modified Eagle medium (Invitrogen) supplemented with 10% (v/v) fetal bovine serum (FBS; Invitrogen). A stable B16F10 cell line expressing membrane-bound OVA (B16F10-OVA) was produced by transient transfection with pCL-neo-mOVA (Addgene, Cambridge, MA) using Lipofectamine 2000 reagent (Invitrogen) and selected with G418 (InvivoGen; San Diego, CA, USA). Naïve CD3+ T cells were purified from mouse spleen and lymph nodes by negative selection using a T-cell enrichment column (R&D Systems). Naïve CD4+ and CD8+ T cells and CD11C+ dendritic cells (DCs) were purified from mouse spleen and lymph nodes by negative selection using an EasySep magnetic separation system (Stemcell Technologies; Vancouver, Canada). To generate mouse T-cell blasts, isolated T cells were incubated in 2 µg/ml anti-CD3/28-coated culture plates with 100 U/ml rIL-2 for 48 h and cultured for an additional 3 days with 100 U/ml rIL-2. The purity of each population was confirmed to be more than 95% by flow cytometry.
3
Isolation and Characterization of Mouse Immune Cells
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Mouse thymocytes were purified from the mouse thymus. Mouse splenocytes and lymphocytes were dispersed and purified to CD4+, CD8+, CD19+ and CD11c+ populations by MACS cell separation (Miltenyi Biotec). Naïve CD3+ T cells were purified from the mouse spleen and lymph nodes by negative selection using a T cell enrichment column (R&D Systems). To generate mouse T cell blasts, naïve CD3+ T cells were incubated in 2 μg/ml anti-CD3/28-coated culture plates with 100 U/ml rIL-2 for 48 h and cultured for a further 5 days with 100 U/ml rIL-2. Mouse splenocytes were dispersed and purified into CD4+, CD8+ and CD19+ populations using the EasySep magnetic separation system (Stemcell Technologies, Vancouver, Canada) or MACS cell separation (Miltenyi Biotec, Bergisch Gladbach, Germany). The purity of each population was confirmed as >95% by flow cytometry. HEK293T cells (CRL-1573, ATCC), B16F10 cells (CRL-6475, ATCC) and EO771 cells (CRL-3461, ATCC) were maintained in DMEM supplemented with 10% FBS, penicillin and streptomycin.
4
Isolation and Analysis of EVs from Stimulated Cells
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WEHI-231 cells were stimulated as described using biotinylated 2° Ab. Following stimulation, the cells were centrifuged at 500 × g for 5 min to pellet cells. Cells were stained for FACS as described below as needed. Supernatant containing vesicles was then centrifuged at 2000 × g for 5 min to pellet cell debris and larger vesicles. Protease and phosphatase inhibitors (1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma-Aldrich, St. Louis MO), 1 mM sodium orthovanadate (Sigma-Aldrich) and 1 µM aprotinin (Sigma-Aldrich)) were added to the supernatant. Anti-CD24 M1/69 (10 µg/mL) and biotinylated 2° Ab (5 µg/mL) were added to supernatant from istotype-treated cells. Supernatant (1 ml) was then incubated with 2.2 × 106 streptavidin-coated magnetic beads (average diameter 4.0 µm; Spherotech; Chicago IL) pre-blocked in 5% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) with rotation, overnight at 4 °C. Beads and the bound EVs were then isolated using an EasySep magnetic separation system (StemCell; Vancouver, Canada) followed by FACS analysis (see below).
5
Generating Antigen-Specific T Cell Subsets
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B16F10 (CRL-6475) cell lines were purchased from ATCC. A stable B16F10 cell line expressing membrane-bound OVA (OVA+B16F10) was produced by transient transfection with pCL-neo-mOVA (Addgene, Cambridge, MA) using Lipofectamine 2000 reagent (Invitrogen) and selection with G418 (InvivoGen, San Diego, CA, USA). For BMDCs cultures, 5 × 106 BM cells were cultured in 10 mL of RPMI supplemented with 20 ng/mL recombinant murine GM-CSF for 7 to 9 days. GM-CSF was added every 3 days. To generate cDC1s, 3 × 106 BM cells were incubated in 3 mL of RPMI supplemented with 200 ng/ mL Flt3-L for 9 days. Flt3-L was added every 2 days and cDC1s (CD11c+B220−) were isolated by anti-B220 positive selection beads to exclude plasmacytoid DCs (CD11C+B220+) for further experiments. However, unless otherwise indicated (for Additional file 1 : Figs. S1 and 2), we used GM-CSF-induced BMDCs for most of the experiments. Naive CD4+ T cells were purified from the mouse spleen and LNs by negative selection using an EasySep magnetic separation system (Stemcell Technologies, Vancouver, Canada). To generate mouse T cell blasts, OTII CD4+ T cells were incubated in 2 µg/mL anti-CD3/28-coated culture plates with 100 U/mL rIL-2 for 48 h and cultured further for 3 days with 100 U/mL rIL-2.
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