Cell subpopulations of CD4+ T cells (CD3+CD4+) and monocytes (CD3−CD14+) were isolated using EasySep magnets (STEMCELL Technologies, Vancouver, British Columbia, Canada). First, PBMCs were washed in FACS buffer (PBS + 2% FBS + 2 mmol/l EDTA) and treated with DNAse (100 ug/ml) (StemCell, #7900) for 15 min at room temperature. Subsequently, Easysep Human CD4+ positive selection kit II (StemCell, #17852) was used according to the manufacturer's protocol, and the flowthrough used for Easysep Human monocyte isolation kit (StemCell, #19359), according to the manufacturer's protocol.
Easysep human cd4 positive selection kit 2
Manufactured by STEMCELL
Sourced in Canada
The EasySep Human CD4 Positive Selection Kit II is a laboratory tool designed for the isolation and enrichment of human CD4-positive T cells from a variety of sample types. The kit utilizes magnetic particles and a specialized buffer system to selectively bind and separate the target cells from the original sample.
Automatically generated - may contain errors
Lab products found in correlation
Dnase, by STEMCELL (1 mentions)
Easysep magnet, by STEMCELL (1 mentions)
Easysep human monocyte isolation kit, by STEMCELL (1 mentions)
Easysep human t cell enrichment kit, by STEMCELL (1 mentions)
Easysep human cd8 positive selection kit 2, by STEMCELL (1 mentions)
Easysep human cd19 positive selection kit 2, by STEMCELL (1 mentions)
Anti human cd4 percp cy5, by BD (1 mentions)
Lymphoprep reagent, by Axis-Shield (1 mentions)
6 protocols using easysep human cd4 positive selection kit 2
1
Isolation of CD4+ T cells and Monocytes
2
PBMC T Cell Enrichment and Activation
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Following the isolation of PBMCs, the T cells were enriched with an EasySep Human T Cell Enrichment Kit (STEMCELL Technologies, Vancouver, BC, Canada). CD8+ T cells were then selected using an EasySep Human CD4 Positive Selection Kit II (STEMCELL Technologies, Vancouver, BC, Canada). The cells were then labeled with CFSE or a CellTrace Violet Proliferation Kit. The CD8+ T cells were then mixed with Dynbeads Human T-Activator CD3/CD28 (ThermoFisher Scientific, Waltham, MA, USA) at a 1:1 ratio in a U-bottom 96-well plate. The cells were monitored for activation and proliferation by flow cytometry.
3
Enrichment of HIV-Infected Cells
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Enrichment of HIV‐infected cells (HIV‐1 p24 positive, CD4 negative) was conducted based on previously described protocols (Davis et al, 2011 ). In brief, HIV‐1‐exposed CD4 T cells were incubated with 25 U/ml Benzonase Nuclease (Merck Millipore Novagen) for 30 min at 37°C, 5% (v/v) CO2, and then washed. Infected cells were subsequently enriched through depletion of CD4‐expressing T cells using EasySep Human CD4 Positive Selection Kit II (Stemcell). CD4(−) cells in the supernatant were collected and used for downstream applications.
4
Isolation of CD8+ and CD4+ T Cells
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PBMCs were isolated, as previously described. CD8+ cells were then isolated from PBMCs using anti‐CD8 magnetic beads (EasySep Human CD8 Positive Selection Kit II; Stemcell Technologies), and CD4+ cells were sequentially isolated from the CD8+‐depleted PBMC pour‐off using anti‐CD4 magnetic beads (EasySep Human CD4 Positive Selection Kit II; Stemcell Technologies).
5
Isolation and Purification of CD4+ T Cells and CD19+ B Cells
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Human whole blood was collected in heparin-treated tubes and peripheral blood mononuclear cells (PBMCs) were isolated by the density gradient centrifugation using LymphoprepTM reagent (Axis-shield, Oslo, Norway). CD4+ T cells and CD19+ B cells were isolated by magnetic microbeads according to the manufacturer’s instructions (EasySep™ Human CD4 Positive Selection Kit II and EasySep™ Human CD19 Positive Selection Kit II, STEMCELL Technologies, Vancouver, Canada). Briefly, human PBMCs were mixed with antibody cocktail conjugated with biotin followed by the incubation with anti-biotin microbeads. The labeled cell mixtures were incubated in a EasySep™ magnet for 3 min. The unbound cells were carefully pipetted from the supernatants and discarded. The tubes containing CD4+ T cells or CD19+ B cells were removed from the magnet and washed once with Magnetic Cell Sorting (MACS) buffer. The purity of CD4+ T cells and CD19+ B cells were detected by flow cytometry using anti-human CD4-Percp-Cy5.5 and anti-human CD19-BV605 antibodies (Abs) (both from BD Biosciences, San Diego, CA, USA), respectively.
6
CD4+ Memory Cell Viral Outgrowth Assay
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