Ficoll hypaque density gradient centrifugation

The study was conducted on cohorts, including 27 HIV-1 infected patients, 12 pulmonary tuberculosis patients (PTB), 8 HIV-PTB co-infected patients (HIV-PTB), and 20 healthy controls (HC). HIV-1 infected patients, confirmed positive by three serologic tests as per National AIDS Control Organization (NACO, Govt. of India) guidelines, were enrolled from the Integrated Counselling and Testing Centre (ICTC) in the Department of Immunopathology, PGIMER Chandigarh, India. At the time of recruitment, the patients were interviewed by a counsellor to obtain informed consent and ascertain therapy naïve status. The status of the disease was assessed by absolute CD4 cell count monitored by flow cytometry using BD Tritest containing antibody conjugates CD3-FITC/CD4-PE/CD45-PerCP with BD Trucount tubes (BD Biosciences, San Jose, USA). PTB and HIV-PTB patients, confirmed positive for Mtb infection by chest x-ray and sputum smear positivity were recruited from DOTS (Directly observed therapy-short course) centre at our hospital. The peripheral blood mononuclear cells (PBMCs) were isolated from the heparinized blood by Ficoll-Hypaque density gradient centrifugation (HiMedia, Mumbai, India).

Confirmed RA patients on disease-modifying anti-rheumatic drugs (DMARDs) and still showing disease activity score (DAS28) 3.2 and above were recruited in the study. The peripheral blood and SF samples of patients (n=15) were collected in a paired manner after informed consent. Mononuclear cells (MNCs) were isolated from heparinized blood and SF by Ficoll-Hypaque density gradient centrifugation (HiMedia, Mumbai, India) and labeled with carboxyfluorescein succinimidyl ester (CFSE). For labeling, 5-6*10⁶ cells were suspended in pre-warmed PBS containing 0.1% bovine serum albumin and 5mM CFSE. After incubating cells at 37°C for 10 min in the dark, cells were washed 2-3 times with cold plain RPMI 1640.
For co-culture, 1*10⁴ UC-MSCs were seeded per well in complete RPMI 1640 medium (containing 10% FBS, 1% Penicillin-Streptomycin, and 2 mM L-Glutamine) along with 1*10⁵ MNCs from SF or peripheral blood (1:10 ratio) in the presence of 4 ug/ml of phytohaemagglutinin (PHA) for 72 h [19 (link)]. The CFSE dye dilution and expression of activation markers (CD25, CD38, and HLA-DR) on MNCs were evaluated by flow cytometry using FACS Diva™ Software (BD, Biosciences).