Fifty milliliters of blood samples were taken from all study patients. Briefly, fresh PBMCs were obtained and rCD4+ T cells were purified by negative selection with EasySep™ Human Resting CD4+ T-Cell Isolation Kit (Stemcell Technologies, Vancouver, Canada), as described previously by our group [9 (link)]. The purity of CD4+CD25-CD69-HLA/DR-T cells was assessed by flow cytometry as >99%. Total RNA was extracted using AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany) from both rCD4 T+ cells and rCD4 T- PBMCs, following manufacturer’s instructions. RNA was quantified with Nanodrop 1000 (Thermo Fisher Scientific, Waltham, MA, USA).
Easysep human resting cd4 t cell isolation kit
Manufactured by STEMCELL
Sourced in Canada
The EasySep Human Resting CD4+ T Cell Isolation Kit is a product designed to isolate resting CD4+ T cells from human peripheral blood mononuclear cells (PBMCs) or whole blood. The kit utilizes a negative selection approach to enrich for the target cell population without the need for columns or magnets.
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Lab products found in correlation
Easysep cd4 t cell enrichment kit, by STEMCELL (2 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Allprep dna rna mini kit, by Qiagen (1 mentions)
Ficoll paque plus, by GE Healthcare (1 mentions)
Primary human umbilical vein endothelial cells, by Lonza (1 mentions)
Sepmate, by STEMCELL (1 mentions)
Lymphoprep, by STEMCELL (1 mentions)
Panobinostat, by Selleck Chemicals (1 mentions)
Vorinostat, by Selleck Chemicals (1 mentions)
Romidepsin, by Selleck Chemicals (1 mentions)
Raltegravir, by Selleck Chemicals (1 mentions)
Interleukin 2 (il 2), by Merck Group (1 mentions)
Pen strep glut, by Thermo Fisher Scientific (1 mentions)
Easysep human cd4 t cell isolation kit, by STEMCELL (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Egm 2 complete culture medium, by Lonza (1 mentions)
5 protocols using easysep human resting cd4 t cell isolation kit
1
Isolation and Characterization of Resting CD4+ T Cells
2
Isolation and Differentiation of Immune Cells
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Peripheral blood mononuclear cells were purified by centrifugation at 1000g during 20 min at room temperature on Ficoll Paque Plus (GE Healthcare). The PBMC layer was collected and underwent 3 washes in PBS (5min centrifugations at 600 g at room temperature).
Resting CD4+ T cell (CD4+CD69−CD25−HLA-DR-) were isolated by magnetic bead separation (EasySep Human Resting CD4+ T cell Isolation Kit, Stemcell), according to the manufacturer protocol.
For MDM differentiation, PBMC were seeded at a concentration of 1.107cells/mL and incubated at 37°C during 3h. Adherent cells were recovered and seeded at a concentration of 2.105cells/mL in presence of 50 ng/mL of M-CSF during 7 days.
Resting CD4+ T cell (CD4+CD69−CD25−HLA-DR-) were isolated by magnetic bead separation (EasySep Human Resting CD4+ T cell Isolation Kit, Stemcell), according to the manufacturer protocol.
For MDM differentiation, PBMC were seeded at a concentration of 1.107cells/mL and incubated at 37°C during 3h. Adherent cells were recovered and seeded at a concentration of 2.105cells/mL in presence of 50 ng/mL of M-CSF during 7 days.
3
Isolation and Culture of PBMC, CD4+ T Cells, and Endothelial Cells
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Peripheral blood mononuclear cells (PBMC) were isolated from EDTA-whole blood by density gradient centrifugation (n = 12) using Lymphoprep and SepMate tubes (StemCell Technologies). Resting CD4+ T cells (rCD4s) were isolated from PBMC by magnetic negative selection using the EasySep Human Resting CD4+ T cell Isolation Kit (StemCell Technologies). In some experiments, bulk CD4+ T cells were isolated using the EasySep CD4+ T cell Enrichment Kit (StemCell Technologies). Isolated CD4+ T cells were maintained in RPMI medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Thermo Fisher, Burlington, Canada). Purity of isolated cells was assessed by flow cytometry.
Primary human umbilical vein endothelial cells (HUVEC) and human dermal microvascular lymphatic endothelial cells (LECs) were obtained from Lonza (Walkersville, MD) and cultured in Lonza EGM-2 or EGM-2MV complete culture media, respectively (Cedarlane, Burlington, Canada). ECs were seeded at 5,000 cells/cm2 and cultured to at least 70% confluence before coculture with CD4+ T cells.
Primary human umbilical vein endothelial cells (HUVEC) and human dermal microvascular lymphatic endothelial cells (LECs) were obtained from Lonza (Walkersville, MD) and cultured in Lonza EGM-2 or EGM-2MV complete culture media, respectively (Cedarlane, Burlington, Canada). ECs were seeded at 5,000 cells/cm2 and cultured to at least 70% confluence before coculture with CD4+ T cells.
4
Activating Latent HIV in CD4+ T Cells
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PBMCs were thawed and either total or resting CD4+ T cells were isolated by negative selection using the EasySep™ Human CD4+ T Cell Isolation Kit or the EasySep™ Human Resting CD4+ T Cell Isolation Kit, respectively (StemCell Technologies, cat # 17952 and 17962, respectively). Cells were cultured at 5 million cells per well in a 24-well plate in RPMI-1640 (Life Technologies, cat # 21870092) supplemented with 10% FBS (Interpath) and pen/strep/glut (Life Technologies, cat # 10378016). Cells were stimulated with DMSO (1:2000, Sigma Aldrich, cat # D2650-5 × 10 ML), vorinostat (500 nM, Selleckchem, cat # S1047), romidepsin (40 nM, Selleckchem, cat # S3020), panobinostat (30 nM, Selleckchem, cat # S1030), JQ1 (1 µM, Selleckchem, cat # S7110), or PMA+PHA (10 nM, Sigma Aldrich, cat # P8139-1 MG; 10 μg/mL, Thermo Fisher Scientific, cat # R30852701), all in the presence of raltegravir (1 µM, Selleckchem, cat # S2005) and IL-2 (1 U/mL, Sigma Aldrich, cat # 10799068001) for 3 days. For the panobinostat study, frozen PBMCs were thawed and total CD4+ T cells were isolated via negative selection using the CD4+ T Cell Isolation Kit, Human (Miltenyi Biotec, cat # 130-096-533).
5
Isolation and Co-culture of PBMC-derived CD4+ T Cells with ECs
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Peripheral blood mononuclear cells (PBMC) were isolated from whole blood by density gradient centrifugation. Resting CD4+ T cells (rCD4s) were isolated from PBMC by magnetic negative selection using the EasySep Human Resting CD4+ T cell Isolation Kit (StemCell Technologies).
In some experiments, bulk CD4+ T cells were isolated using the EasySep CD4+ T cell Enrichment Kit (StemCell Technologies). Isolated CD4+ T cells were maintained in RPMI medium supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin (R10 medium).
Purity of isolated cells was assessed by flow cytometry.
Human umbilical vein endothelial cells (ECs) were obtained from Lonza and cultured in EGM-2 complete culture medium (Lonza). ECs were seeded at 5000 cells/cm 2 and cultured to at least 70% confluence before co-culture with CD4+ T cells.
In some experiments, bulk CD4+ T cells were isolated using the EasySep CD4+ T cell Enrichment Kit (StemCell Technologies). Isolated CD4+ T cells were maintained in RPMI medium supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin (R10 medium).
Purity of isolated cells was assessed by flow cytometry.
Human umbilical vein endothelial cells (ECs) were obtained from Lonza and cultured in EGM-2 complete culture medium (Lonza). ECs were seeded at 5000 cells/cm 2 and cultured to at least 70% confluence before co-culture with CD4+ T cells.
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