Rosettesep human cd4 t cell enrichment cocktail

CD4⁺ T cells were obtained by negative selection from peripheral blood of healthy controls (HCs) (N = 4; 3 female and 1 male; average age of 42) following informed consent in accordance with National Health Service Research Ethic Committee approved protocols at the University Hospitals Bristol Foundation Trust, United Kingdom (04/Q2002/84). Written, informed consent was obtained from all study participants. CD4⁺ T cells were obtained by incubating up to 80 ml uncoagulated peripheral blood with RosetteSep™ Human CD4⁺ T cell Enrichment Cocktail (Stemcell Technologies, Canada) according to the manufacturer's instructions. Blood was then layered on Ficoll-Paque PLUS (GE Healthcare, USA) and centrifuged for 1,200 × g for 20 min. Enriched CD4⁺ T cells were removed from the density gradient and washed in RPMI-1640 (Thermofisher) supplemented with 10% fetal calf serum (FCS). The purity of human CD4⁺ T cells was >95%.

HEK293T (ATCC, Cat# CRL-3216) and TZM-bl (NIH, Cat# 8129) cells were cultured in Dulbecco modified eagle medium (DMEM) supplemented with 10% (v/v) FBS, 2 mM L-glutamine, 100 µg/ml streptomycin and 100 U/ml penicillin (all from Gibco). MOLT-4 clone 8 (NIH, Cat# 175) and THP-1 (ATCC, Cat# TIB-202) cells were cultured in supplemented Roswell Park Memorial Institute (RPMI) 1640 medium. All cells grew at 37 °C with 5% CO₂ and were tested routinely for mycoplasma contamination by a PCR-based method. The cell lines were authenticated by ATCC or NIH and not validated further in our laboratory.
PBMCs from healthy human donors were obtained from lymphocyte concentrates from 500 ml whole blood (blood bank Ulm) and PBMCs from fresh blood of healthy AGMs (Silabe, Strasbourg) by density centrifugation. For viral infection, cells were pre-stimulated with 1 μg/ml phytohemagglutinin (PHA) and 10 ng/ml human recombinant interleukin-2 (IL-2) for 3 days. Human CD4+ T cells were enriched from buffy coats through negative selection using the RosetteSep™ Human CD4+ T Cell Enrichment Cocktail (Stemcell) according to manufacturer’s protocol and stimulated with 10 ng/ml IL-2 and Dynabeads® Human T-Activator CD3/CD28 in a ratio of 1:1.

To isolate CD4+ T-cells, PBMCs from 5 CMV-positive and 5 CMV-negative CHF patients were isolated by centrifugation on Ficoll-Hypaque gradients (Lymphoprep; Nycomed, Oslo, Norway) after 20 min of incubation with the RosetteSep Human CD4+ T-cell Enrichment Cocktail (StemCell Technologies, Grenoble, France). In all cases, purity of isolated CD4+ T-lymphocytes, tested by flow cytometry was higher than 95%. mRNA was extracted using a Total RNA Isolation (Macherey-Nagel GmbH & CoKG, Düren, Germany) according to the manufacturer’s instructions. Reverse transcription of mRNA isolated from each sample was carried out in a 20 µL final volume with the iScript cDNA Synthesis Kit (Bio-Rad, Life Science Research Group, Hercules, CA, USA) following manufacturer’s instructions. The mixture was incubated at 25°C for 5 min, at 42°C for 30 min, and at 85°C for 5 min and stored at -80°C until required for the array. Equal quantities of cDNA were mixed to generate two pools, one with samples from CMV-seronegative patients and another one with samples from CMV-seropositive patients. Cytokine gene expression was examined through TaqMan™ Array Human Immune Response Real-Time PCR (Applied Biosystems, Foster City, CA, USA) using predesigned human gene-specific primers and probes based on published cytokine sequences and following manufacturer’s instructions.

Pulp tissue was extracted and digested with 200u/ml collagenase II (Worthington) at 37°C for 1 hour. Bone marrow mononuclear cell fractions were purchased from Lonza, (n = 9). Both bone marrow MSCs (bmMSCs) and dental pulp MSCs (dpMSCs) were cultured in 10% DMEM at 37°c 5%CO_2. Peripheral blood mononuclear cells (PBMCs) from healthy donors were obtained from anonymized leukocyte cones supplied by the National Blood Transfusion Service (NHS Blood and Transplantation, Tooting, London, UK). PBMCs were isolated by Lymphocyte (PAA, Austria) density gradient. RosetteSep Human CD4⁺ T cell enrichment cocktail (STEM CELL, Cambridge, UK) was used to obtain purified CD4⁺ T cells. The purity of CD4⁺ T cells was between 95–99%. Mouse bmMSCs were isolated from hind limb tibia of C57BL/6j mice and cultured in alphaMEM + 20% FCS at 37°c 5%CO₂. Mouse CD4⁺ T-cells cells were isolated from solenocytes by negative depletion using mouse CD4⁺ T-cell isolation kit (Miltenyi). C57BL/6j were maintained under pathogen-specific sterile conditions in the Biological Services Unit at King’s College London. All procedures were performed in accordance with institutional guidelines and the Home Office Animals Scientific Procedures Act (1986).

THP-1 cells were grown in RPMI 1640 GlutaMAX (Gibco) supplemented with 10% heat-inactivated fetal calf serum (FCS) and 100 U/ml penicillin and 100 μg/ml streptomycin. THP-1 cells were differentiated with 25 ng/ml phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich) for 24 h. The purification of primary blood mononuclear cells has been described before (70 (link)). Primary CD4⁺ T cells or CD14⁺ monocytes were derived from 50 ml of whole blood or from buffy coats and grown in RPMI 1640 medium with GlutaMAX with 10% heat-inactivated FCS and penicillin-streptomycin. CD4⁺ T cells were isolated using the CD4⁺ T cell isolation kit II (Miltenyi Biotec) or the RosetteSep human CD4⁺ T cell enrichment cocktail (Stem Cell Technologies), activated with 100 IU/ml interleukin-2 (IL-2; Biomol) and 2 μg/ml phytohemagglutinin (PHA; Sigma-Aldrich) for 3 days. MDMs were differentiated for 7 days using 100 ng/ml granulocyte-macrophage colony-stimulating factor (R&D Systems). 293T were grown in Dulbecco modified Eagle medium (DMEM-GlutaMAX; Gibco) with 10% heat-inactivated FCS and penicillin-streptomycin.

CD4⁺CD45RA⁺ T cells were isolated from whole blood from healthy donors. For mononuclear cell isolation, density gradient centrifugation by Ficoll–Hypaque (Merck KGaA, Darmstadt, Germany) was performed. CD4⁺ T cells were isolated by RosetteSep™ Human CD4⁺ T Cell Enrichment Cocktail (Stem Cell Technologies, Vancouver, Canada). CD4⁺CD45RA⁺ T cells were then isolated by negative selection using an EasySep™ Human Naїve CD4⁺ T Cell Isolation Kit (Stem Cell Technologies, Vancouver, Canada). The purity of CD4⁺CD45RA⁺ T cells was greater than 92%.