HeLa cell (1 × 105 cells/ml) were maintained in minimal essential medium (MEM, Gibco BRL, Tokyo Japan) supplemented with 10% fetal bovine serum and 100,000 U/1 penicillin at 37°C in humidified atmosphere with 5% CO2. The cultured medium was replaced every 3 days. Cells were rinsed 2 times with phosphates-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 8.1 mM NaH2PO4) before addition of fresh medium.
Bacillus Calmette-Guérin(BCG) [7, 1, 29]
Mycobacterium bovis Bacillus Calmette-Guérin(BCG) Tokyo was cultured in Middle brock 7H9 broth (Difco Laboratories, Detroit, MI, USA) supplemented with 10% Albumin-dextrose-catalase(ADC; Difco laboratories) enrichment and 0.1% Tween 80. The cells(approximate 8 × 1010 cells/ml) were harvested with shaking at 37°C until 0.8 of an optical density(OD) at 590 nm. It was centrifuged and the pelletized cells re-suspended with MEM were divided and used for experiments. Dead BCG dosage (1 × 107 cells/ml) as MOI 100 was prepared and treated by an autoclave (121°C, 10 min).
Fraction of BCG membrane and cytoplasm 
BCG(Tokyo) was sedimented (3,000 × g, 10 min, 4°C), suspended in TMNSH buffer, and lysed by sonication in a Bioruptor UCD-200T sonicator (Toso, Tokyo, Japan). The cell lysate was centrifuged at 10,000 × g for 10 min at 4°C twice. The supernatant was then centrifuged at 30,000 × g for 30 min at 4°C. The pellet was used as the membrane fraction.
The supernatant solution was centrifuged under the same conditions, and the supernatant thus obtained was then centrifuged at 105,000 × g for two hours at 4°C. The pellet was used as the cytoplasm fraction.
The pellets obtained in each step were suspended in TMNSH buffer. Freeze drying membrane and cytoplasm fraction were prepared 0.04 μg/ml and 0.02 μg/ml respectively for the experiment as MOI 100 and keeping at the department of oral bacteriology Nagasaki University. BCG also had been cultured in the same department.
Polyclonal antibody of MPB70 (secreted protein, α antigen) [5, 6, 10]
Purified MPB70 was provided by Dr. Nagai. BALB/c mice at 7-10 weeks of age were immunized intravenously with MPB 70 (10 μg diluted 200 ul of PBS) which was served as the most abundant protein in the culture filtrate from BCG(Tokyo). After 30 days, same amount of MPB 70 was injected intraperitoneally to boost the immune response. After 1 week later, sera were collected from the eye vein of immunized mice and pooled at -80°C until use. Animal had been keeping at animal center of Nagasaki Univ.
HeLa and BCG cells co-culture
Two millimeter of HeLa cells (1 × 105/ml) were inoculated in 24-well plates and cultured for 1 days. Then BCG at various doses and type were added to the wells. After 72 hours fresh MEM exchanged one ml.
Different BCG dosage even, ten and hundred times of multiplicity of infection (MOI) BCG were prepared and cultured with HeLa cell.
Growth inhibition of HeLa cell checked by its fraction of membrane (0.04 μg/ml, Dry weight) and cytoplasm (0.02 μg/ml, Dry weight) were also prepared as equivalent dosage of MOI 100 and added in the medium of HeLa cells.
Internalization of the BCG checked using each cytochalasin B (100 μg/ml, Wako pure C.I. Japan) or heparin sodium (0.001 U/ml, OSTUKA Pharm. Japan) was added into the well before co-cultured with HeLa cell respectively.
Every other day during the incubation period with the HeLa and BCG, MEM changed to the usual saline solution and treated 2% tripsin treated for 5 minutes. HeLa cells were mounted on the erythrocytometer after 0.3% trypanblue stain. The number of cells expressed as the mean for three times.
Transmission electron microscope (TEM) 
The HeLa cell washed in the normal saline solution and centrifuged. The cells were fixed in solution of 2% glutaraldehyde in 0.1 M phosphate buffer solution, pH 7.3 for 2 hours and 1% osmium tetraoxide for 2 hours. After two times washed PBS and water the cells were dehydrated with increasing concentrations of ethanol; and gradually infiltrated with Epon 812. Before inspection by TEM the trimmed bloc of epon was orientated and stained with toluidine blue for light microscopy orientation. The ultrathin section with silver to gold interference color were picked up in a nickel grid and stained with uranyl acetate and lead nitrate in the usual manner.
For Immuno-TEM HeLa cell fixed 1% paraformaldehyde at 4°C for one hour. Then dehydrated in ethanol and embedded by LR white(Okenshoji, Japan) for 2 days at -20°C. The same interference color picked up on the collodion coated mesh (Nissinn EM, Japan) specimens were reacted with 2% hydrogen peroxide for 30 minutes twice and blocked bovine serum for 30 minutes. They were reacted with hundred times diluted anti-sera (polyclonal Aanti-MPB70) for 12 hours at 8°C and protein A gold (15 nm, FUNAKOSI, Japan) for three hours at room temperature in moisture chamber and then double stained for 5 minutes each. The specimens were examined in a H800 electron microscope (Hitachi, Japan) operating at 75 kV.