Easysep human memory cd4 t cell enrichment kit

Mem were purified using the untouched memory CD4 isolation kit (EasySep human memory CD4⁺ T-cell Enrichment Kit; StemCell Technologies) allowing for more than 94.6% purification without any cell stimulation and apoptosis.

Cryopreserved PBMCs from 1 of the 27 individuals (patient 107) were thawed and cultured in RPMI 1640 supplemental with 2 mM Glutamax (both from Gibco), 10% human serum (MilliporeSigma), and 100 U/ml penicillin and 100 μg/ml streptomycin (Thermo Fisher) (complete RPMI), with 50 μg/ml natural Ara h 1 (Indoor Biotechnologies), at a density of 6 × 10⁶ cells in 1 ml medium per well in 24-well plates. Complete RPMI + 10 U/mL IL-2 (R&D Systems) was added after 5 days, and cells were cultured for a total of 14 days to expand Ara h 1–specific CD4⁺ T cells. After harvesting, memory CD4⁺ T cells were isolated with the EasySep human memory CD4⁺ T cell enrichment kit (Stemcell Technologies) and labeled with APC-conjugated Ara h 1 (DRB1*03:01, amino acid 415–425) tetramer (32 (link)) (made in-house), at a concentration of 10 nM for 1 hour at room temperature. After washing off excess tetramer, the cells were labeled with BUV395-conjugated anti-CD3 (clone UCHT1; BD Biosciences), APC-Cy7–conjugated anti-CD4, FITC-conjugated anti-CD45RA, and Live/Dead Fixable Blue stain (L23105; Thermo Fisher) for 30 minutes at 4°C. Live CD3⁺CD4⁺CD45RA^– tetramer⁺ and tetramer^– T cells were sorted with a FACSAria Fusion instrument (BD Biosciences), and genomic DNA was isolated using the AllPrep DNA/RNA Micro Kit.

PBMCs and LN mononuclear cells (LNMCs) were isolated using Ficoll–Paque density centrifugation. PBMCs and LNMCs were viably frozen in 90–95% fetal bovine serum and 5–10% dimethylsulfoxide. For analysis, cells were thawed and subjected to negative immunomagnetic isolation of memory CD4⁺ T cells, using a commercial product (Stemcell EasySep Human Memory CD4⁺ T cell Enrichment Kit, no. 18000) per the manufacturer’s protocol.

CD4⁺CD45RO⁺ T cells were isolated from PBMC using the immunomagnetic negative selection EasySep Human Memory CD4⁺ T Cell Enrichment Kit and Magnet (StemCell Technologies, Vancouver, Canada), following manufacturer provided indications. Recovery (40.9±14.1%) and purity (93.6±1.2%) of the enriched population was assessed by flow cytometry, staining with PE-Cy7 anti-CD4 (BioLegend, San Diego, CA, USA), FITC anti-CD3, PE-Cy5 anti-CD8, APC anti-CD45RA and PE anti-CD45RO (BD Biosciences, San Diego, CA, USA) antibodies.
For initial stimulation, memory CD4⁺ T cells were seeded in 96-well plates, in T cell medium, at 5×10³ cells/well and incubated with 10⁴ autologous irradiated PBMC and 2.5 μg/ml T. cruzi epimastigote lysate, or at 10³ cells/well and incubated with 10⁴ allogeneic irradiated PBMC from 3 non-related, non-infected donors, 1 μg/ml PHA and 50 IU/ml IL-2. This interleukin was also added on every culture since day 3, every 3 to 4 days, at a final concentration of 50 IU/ml. Half the medium (100 μl/well) was refreshed every 10 to 14 days, beginning at day 15.

Memory CD4⁺ T cells were isolated from three different HIV-1-infected donors who were receiving HAART and had undetectable levels of viral RNA with the EasySep Human Memory CD4⁺ T-cell Enrichment kit (Stem Cell catalog no. 19157). One million cells were treated with 100 nM GSK-343, EPZ-6438, or UNC-0638 for 72 h. Cells were left untreated or further treated with IL-15 (50 ng/ml) or SAHA (500 nM) overnight. Total RNA was isolated and subjected to reverse transcription (RT)-PCR with primers that specifically amplified singly spliced env mRNA of HIV-1. Next-generation sequencing of RT-PCR products was performed to measure the abundance of env mRNAs. The number of reads was converted into the equivalent number of cells harboring HIV-1 per 10⁶ cells by using a standard curve prepared from HIV-infected cells sorted by flow cytometry.