Ficoll paque premium

Fourteen-week-old, male, specific pathogen-free New Zealand White (052 CR; 571 OAKWOOD) rabbits were obtained from Charles River Laboratories (Wilmington, MA). After a 2-week acclimatization period, 1 x 10⁷ lethally irradiated (100 Gy) 729.B control cells or 729 HTLV-1 producer cells (wt or mEnhancer) were inoculated into the lateral ear vein. Blood was drawn via the central auricular artery at Weeks 0 (pre-inoculation), 4, 8, 12, 16, 20, and 25 (study endpoint). Plasma was collected and rabbit PBMCs (rPBMCs) were isolated using Ficoll-Paque™ PREMIUM (Cytiva, Marlborough, MA). rPBMCs or plasma were assessed for proviral load, HTLV-1 gene expression, and HTLV-1-specific antibody response, as described below. Sanger sequencing of the viral enhancer region was performed at week 25 to monitor for viral reversions. All animal procedures were performed in accordance with a protocol approved by University Laboratory Animal Resources (ULAR) of The Ohio State University.

We obtained peripheral blood from the patients who visited the emergency department within 6 h after the onset of pain; blood samples from patients with plaque rupture identified by OCT examination were used for subsequent analysis, and patients without AMI detected by CAG examination served as the control group. The whole blood samples were collected in ethylenediaminetetraacetic acid (EDTA) tubes before heparin or any contrast agent was administrated. Peripheral blood mononuclear cells (PBMCs) were isolated from all blood samples within 2 h of collection using Ficoll-Paque PREMIUM (Cytiva) according to the manufacturer's instructions.

A density gradient media 1.084 g/mL (Ficoll-Paque^™ PREMIUM, Cytiva, Marlborough, MA, USA) was used to isolate mononuclear cells from 2 mL of anticoagulated blood obtained at 5 and 12 dpi, according to the manufacturer’s instructions. For each hen, 10⁶ cells of the obtained peripheral blood mononuclear cells (PBMCs) were washed in a PBS containing 1% bovine serum albumin (BSA) (Sigma-Aldrich, Saint Louis, MO, USA) followed by centrifugation at 211 xg for 5 min at 4 °C. The cells were resuspended in 0.2% chicken serum (diluted in 1% BSA) and incubated for 15 min at 4 °C for Fc blocking. After centrifugating the cells as indicated above, the supernatant was discarded and the cell pellets were resuspended in the dark for 20 min at 4 °C using fluorescein isothiocyanate (FITC)-conjugated mouse anti-chicken CD8 (Southern Biotech, Birmingham, AL, USA) and phycoerythrin (PE)-conjugated mouse anti-chicken CD4 (Southern Biotech, Birmingham, AL, USA). The stained PBMCs were washed twice with 1% BSA before being fixed in 1% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA). The samples were analyzed at the Flow Cytometry Core Facility, University of Calgary (BD LSR II flow cytometer, BD Bioscience, San Jose, CA, USA). Data acquisition was done using BD FACSDiva^™ 6.1.3 software (BD Bioscience, San Jose, CA, USA).

Peripheral blood mononuclear cells (PBMCs) were isolated from fresh patient-derived EDTA blood. The blood volume was determined and equally diluted for gradient centrifugation with cold RPMI (Gibco, Paisley, GB and Grand Island, NY, USA) without supplements. Diluted blood was then layered carefully on top of 15 mL Ficoll-Paque^® PREMIUM (Cytiva, Marlborough, MA, USA) and centrifuged without a brake at room temperature for 18 min. After centrifugation, the upper layer (plasma) was collected and stored at −80 °C. The PBMC layer underneath was carefully removed from the Ficoll layer by using a 1 mL pipette. PBMCs were washed with RPMI two times, counted, and archived in 1 mL Kryo safe medium I (c.c.pro, Oberdorla, Germany) per tube.

White blood cell count (WBC) and lymphocyte count of whole blood were analyzed using a hematology analyser XP‐300 (Sysmex). Blood was drawn in lithium‐heparinized tubes and processed within 24 h. The peripheral blood mononuclear cells (PBMC) isolation was performed using a density gradient with SepMate tubes according to manufactures instructions (StemCell). Briefly, the blood was mixed 1:1 with phosphate‐buffered saline (PBS) (Biowest) before being applied to the SepMate tubes containing Ficoll‐Paque premium (Cytiva). The tubes were centrifuged at 1200×g for 10 min at room temperature (RT). The top layer was poured off and washed with PBS following centrifugation at 300×g for 8 min at RT. An additional wash was performed with PBS following the last centrifugation of 200×g for 5 min at RT before frozen down in fetal bovine serum (FBS) (Gibco) with 10% dimethyl sulfoxide (DMSO) (Tocris).

Human blood samples involved in this study were approved by the Ethics Committee of Chongqing Public Health Medical Center. Informed consent was obtained from all subjects. Immune non-responder and responder HIV-1 infected patients were recruited at the AIDS Outpatient Department of Geleshan Hospital of Chongqing Public Health Medical Center. The blood samples of healthy individuals came from Chongqing Public Health Medical Center (Pingdingshan hospital area). PBMCs were isolated from fresh whole blood in a 15 ml centrifuge tube using Ficoll-Paque PREMIUM (Cytiva, USA, 17544203) according to the manufacturer’s instructions. After mixing the blood sample with sterile PBS in equal volume, the mixture was slowly added to the Ficoll cell separation media and centrifuged 500 × g horizontally for 15 min at room temperature. Cell numbers and viability were measured using a hemocytometer with Trypan blue staining.