Easysep human cd4 t cell isolation kit

Peripheral venous blood was collected from healthy adult volunteers under ethical agreement code AMREC 15-HV-013. All ethical regulations from the University of Edinburgh were adhered to and informed written consent was obtained from every donor. Experimental programmes were overseen by the University of Edinburgh Centre for Inflammation Research Blood Resource Management Committee. Up to 160 ml of blood was collected at one time into sodium citrate and was then processed immediately. Ficoll Paque Plus (GE Healthcare #GE17-1440-02) was used to isolate mononuclear cells by mixing with freshly isolated blood (1:1 diluted in PBS) and spinning in LeucoSep tubes (Grenier #227289) for 15 min at 1000 g, with brake at 0. CD4⁺ T cells were then isolated from the cell layer using the EasySep^TM human CD4⁺ T cell isolation kit (StemCell Technologies #17952), according to manufacturers’ instructions.

Peripheral blood was obtained from 3 healthy volunteers, two females and 1 male, aged between 25 and 42 years. Written informed consent was obtained from each participant and the protocol was supervised and approved by the Clinical Research Ethics Committee of the Alicante General Hospital (HGUA). Peripheral blood mononuclear cells (PBMCs) were isolated by gradient centrifugation using Biocoll solution (Biochrom, Berlin, Germany). CD4⁺ cells were then isolated from PBMCs by immunomagnetic selection using an EasySep^TM Human CD4⁺ T cell isolation kit (StemCell Technologies, Vancouver, BC, Canada).

CD4+ T cells were isolated from fresh or cryopreserved PBMCs using EasySep™ Human CD4+ T Cell Isolation Kit (#17952, StemCell technologies), after which genomic DNA was extracted using QIAamp DNA Mini Kits (#51304, Qiagen). Total and intact HIV DNA were measured simultaneously with the Rainbow proviral DNA digital PCR assay (38 (link)), using the QIAcuity digital PCR platform. This multiplex assay includes RU5 primers and probes for total HIV DNA detection (76 (link)) and the psi and env primers and probes of the IPDA assay (39 (link)). RPP30 was used as a reference gene (38 (link)). Intactness was quantified based on the detection of env and psi and corrected for shearing using a DNA Shearing Index (DSI) correction, as previously described (39 (link)). Defective HIV DNA was calculated by subtracting intact from total HIV DNA.

Stocks of HIV JR-CSF were prepared as previously described [50 (link)–55 (link)] and standardized by p24 ELISA. Humanized BLT mice were challenged i.v. with HIV JR-CSF (100 ng of p24 or 10⁴ Median Tissue Culture Infectious Doses (TCID₅₀). We used a “simple randomization” for the 4 arms by choosing randomly 8 mice per arm. Three weeks post-HIV challenge, infection was confirmed by quantifying viral RNA by PCR viral load in peripheral blood (plasma) using one-step reverse transcriptase quantitative real-time PCR (qRT-PCR) (ABI custom TaqMan Assays-by-Design) according to the manufacturer’s instructions. Primers were 5-CATGTTTTCAGCATTATCAGAAGGA-3 and 5-TGCTTGATGTCCCCCCACT-3, and MGB-probe 5-FAM-CCACCCCACAAGATTTAAACACCATGCTAA-Q-3, where FAM is 6-carboxyfluorescein as we recently described [51 (link)–52 (link)]. The assay sensitivity was of 423 RNA copies per mL of plasma. For quantification of HIV RNA loads in tissues, RNA was extracted from at least 2x 10⁶ CD4+ cells isolated from the harvested tissues using EasySep^™ Human CD4+ T Cell Isolation Kit (STEMCELL Technologies) and the RNeasy Mini Kit (Quiagen) and viral loads quantified by qRT-PCR as described above.

Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of healthy pregnant women and patients with PE. The isolation was performed using Ficoll–Hypaque with density gradient centrifugation. Venous blood (2 mL) collected from all subjects was mixed with Hank’s solution (2 mL). After adding 2 mL Ficoll–Hypaque solution (MP Biomedicals, Aurora, OH, USA), the samples were centrifuged at 2000 r/min for 5 min. Next, the mononuclear cell layer was transferred to another centrifuge tube, mixed with Hank’s solution, and centrifuged at 2000 r/min for 5 min. After washing with Hank’s solution, PBMCs were maintained in PBMC complete medium (Procell, Wuhan, China) at 37°C with 5% CO₂.
CD4⁺ T cells were isolated from PBMCs using the EasySep™ Human CD4⁺ T Cell Isolation Kit (StemCell Technologies, Vancouver, BC, Canada). Briefly, 5 х 10⁷ cells were incubated with cocktail (50 μL) for 5 min and the samples were mixed with RapidSpheres (50 μL) and the EasySep™ Buffer was added up to 2.5 mL. The samples were then incubated with a magnet for 3 min at 25°C. After removing the magnet, an enriched cell suspension remained.

PBMCs were obtained from the Stanford Blood Bank in California or from the Blood Bank at Aarhus University Hospital in Denmark. PBMCs were purified from buffy coats by Ficoll-Hypaque (GE Healthcare, #17-1440-03) density gradient centrifugation and then cryopreserved in 80% FCS, 10% RPMI10 and 10% DMSO (Sigma, #D4540). PBMCs were thawed 3 days prior to fibroblast co-culture and infection, and were activated using 1 µg/ml plate-bound anti-CD3/CD28 (#16-0037-85 and #16-0289-85, both from eBioscience) in PBS supplemented with 100 U/ml human recombinant IL-2 (PeproTech, #200-02). Where indicated, CD4+ T cells were first purified from PBMCs by immuno-magnetic negative selection with the EasySep™ Human CD4+ T Cell Isolation Kit (Stemcell, #17952).

PBMCs were collected from five HIV-1 seropositive subjects on stable suppressive ART and three healthy HIV-seronegative donors (Supplementary file 1e). All subject signed informed consent forms approved by the Indian Institute of Science, Bangalore and Bangalore Medical College and Research Institute (BMCRI) review boards (Institute human ethics committee [IHEC] No-3-14012020). The PBMCs isolated from blood samples using Histopaque-1077 (Sigma-Aldrich) density gradient centrifugation was used for CD4⁺ T cells purification. The CD4⁺ T cells were purified from 50×10⁶ PBMCs using EasySep Human CD4⁺ T cell isolation kit (STEMCELL Technologies).