K2e tubes

To achieve MR silencing in vivo, doxycycline (Dox) was administered by offering food pellets containing 735 mg/kg doxycycline hyclate (Ssniff Spezialdiäten, Soest, Germany; effective concentration: 625 mg/kg Dox) ad libitum to the mice for at least 14 days. Control animals received a standard diet from the same supplier. Blood was collected by heart puncture after having sacrificed the mice, and serum and plasma were separated using Microtainer SST or K2E tubes, respectively (BD Biosciences, Heidelberg, Germany). In order to perform gene expression and histological analyses, individual organs were dissected and either snap-frozen in liquid nitrogen or fixed in formaldehyde solution.

A total of 80 HHCs of bacteriologically confirmed active pulmonary TB patients (diagnosed either as drug susceptible TB [Cat I], drug susceptible-relapse [Cat II] or Drug resistant TB [Cat IV] cases as per National Guidelines at Lok Nayak Chest Clinic, National TB Elimination Program-New Delhi, Government of India), who were in close contact of more than or equal to 8 h/day for at least 3 months, with the respective index TB patient after onset of the infection, were recruited for the study (Fig. 1). Sample size was estimated based on statistical power of 0.8, medium effect size of 0.6 and significance criterion α = 0.05 using the R package pwr (29 , 30 ). The medium effect size of 0.6 was considered in the case of HHCs as they were at a higher probability of having LTBI than the general population (31 (link)). Further, all the HHCs were screened for TB-disease using chest radiography (CXR) and GeneXpert MTB Rif testing (Cepheid, USA), and only those found to be negative for TB-disease, and having no previous TB-history, were recruited for the study. The exclusion criteria for the recruited HHCs was that they should not have any history of diabetes, smoking and alcohol use. 4 mL of venous blood was collected from the subjects in Lithium Heparin tubes (367886, BD, UK) for IGRA analysis and 500 μL was collected in K2E tubes for RNA extraction (367836, BD, UK).

Mice at ages 4-, 8-, and 13-weeks were fasted for 4 h and then anesthetized (4% isoflurane) during analgesia (IP morphine 0.04 mg in 100 μL sterile water). Blood was retrieved by heart puncture in EDTA-containing K2E tubes (BD, Franklin Lakes, NJ, USA). Tubes were inverted 10 times, immediately put on ice and centrifuged (10 min, 2000 g, 5°C) to retrieve plasma, which was stored at -80°C until analysis. IAA were measured using the Mouse Insulin Autoantibody ELISA kit (Abbexa Ltd, Cambridge, United Kingdom).