*2.1. Study Subjects*

The Committee on Human Rights Related to Research Involving Human Subjects at Walailak University approved all experimental protocols in the present study (approval no. 14/057, 1 August 2014). A total of fourteen Pb-exposed workers (boat caulkers and fishing net workers included) from Pak Phanang district, Nakhon Si Thammarat Province, Thailand, were enrolled in this study. Additional age-matched farmers (*n* = 16) were enrolled as controls. The main criterion for inclusion in the worker group was blood Pb concentration ≥25 μg/dL. For controls, blood Pb levels were <25 μg/dL. The blood Pb level of 25 μg/dL was an exposure limit for Pb-exposed workers, based on the Occupational Safety and Health Administration (OSHA) [24].

#### *2.2. Sampling and Blood Analysis*

From each subject, a 50-mL blood sample was drawn from the median cubital vein in the morning, before working and without fasting. The blood sample of each subject was divided into three aliquots of 5, 10, and 35 mL. The 5-mL aliquots of whole blood, in BD Vacutainer ®EDTA tube (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) were assayed for Pb concentrations, using the graphite furnace atomic absorption spectrometer, AAnalystTM 600 (PerkinElmer, Wellesley, MA, USA), available at Toxicology Laboratory, Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand. Whole Blood Metals Control LymphochekTM Levels 1, 2 and 3 were used for quality assurance and control (Bio-Rad, Hercules, CA, USA). The coe fficients of variation of blood Pb concentrations were within 10%. None of study subjects had blood Pb concentrations below the detection limit of 1.0 μg/dL [25]. The 10-mL aliquots of blood samples contained heparin as an anticoagulant were assayed for T cell subpopulations and phagocytic activity. The 35-mL aliquots of blood samples contained heparin as an anticoagulant were subjected to preparation of mononuclear cells, as detailed in Section 2.3.

#### *2.3. Isolation of Peripheral Blood Mononuclear Cells (PBMCs)*

PBMCs were isolated by the Ficoll density gradient centrifugation method [26] from 35-mL aliquots of blood samples, collected in BD Vacutainer ®Lithium Heparin tubes (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). Cells were counted and cell viability was determined by trypan blue staining. The PBMCs 2 × 10<sup>6</sup> cells/mL were cultured in RPMI 1640 GibcoTM culture medium

(Thermo Fisher, Waltham, MA, USA) supplemented with 15% fetal bovine serum (Merck, Darmstadt, HE, Germany) and 1% penicillin-streptomycin Gibco™ (Thermo Fisher Scientific, MA, USA).

#### *2.4. Phagocytic Activity Assay*

Phagocytosis activity was determined using the IgG-FITC Phagocytosis assay kit (Cayman Chemical, Ann Arbor, MI, USA). The instructions in the manufacturing manual were slightly modified. One milliliter of buffy coat from heparinized blood was mixed with 9 mL FACSTM lysing solution (Becton Dickinson and Company, Franklin Lakes, NJ, USA), and the samples were incubated for 15 min at room temperature. The cells were centrifuged for 5 min at 400× *g* at room temperature. The supernatant was carefully decanted, and cells were adjusted with PBS to a concentration of 1 × 10<sup>6</sup> cells/mL. Cells were transferred into polypropylene FACSTM tubes (Becton Dickinson and Company, Franklin Lakes, NJ, USA) and mixed with the latex beads rabbit IgG-FITC solution. Subsequently, cells were incubated in the dark in an incubator with 5% CO2, 37 ◦C for 1 h. The percentages of active phagocytic cells were determined by BD FACSCaliburTM flow cytometry (Becton Dickinson and Company, Franklin Lakes, NJ, USA).
