Mcfarland standards

CHO expressed recombinant human LF (endotoxin is < 0.2 EU/mg) was kindly provided by PharmaReview Corporation (Houston, TX) [20 (link),36 ].
Mycobacterium bovis, Bacillus Calmette Guerin (BCG), Pasteur strain, (TMC 1011, ATCC, Manassas, VA) was grown in Dubos base (without addition of glycerol) with 10% supplement (5% BSA and 7.5% dextrose in saline) on an orbital shaker at 37 C for 2 weeks before use. BCG was diluted with 1x Dulbecco’s phosphate-buffered saline (PBS) (Cellgro, Herndon, VA) to 3 × 10⁸ organisms/mL, the concentration was estimated using McFarland standards (Sigma, St. Louis, MO), and confirmed by plating dilutions onto 7H11 agar plates (Remel, Lenesa, KS). Plates were incubated at 37 °C with 5% CO₂ for 3–4 weeks, and resulting colonies counted.
Lipopolysaccharide (LPS) from Escherichia coli O111:B4 was purchased from Sigma.

C57BL/6 (female, aged 4–6 weeks) mice were purchased from Jackson Labs (Bar Harbor, ME, USA). All in vivo experiments were conducted under approved guidelines of the animal ethics committee at the University of Texas, Health Science Center at Houston (HSC-AWC-06-100 and HSC-AWC-07-016). Six to 10 mice are included in each experimental group.
Bovine LF (bLF) and CHO expressed recombinant human LF (rHLF) (endotoxin is <0.2 EU/mg) were kindly provided by PharmaReview Corporation (Houston, TX, USA).
Mycobacterium bovis, Bacillus Calmette Guerin (BCG), Pasteur strain, (TMC 1011, ATCC, Manassas, VA, USA) was grown in Dubos base (without addition of glycerol) with 10% supplement (5% BSA and 7.5% dextrose in saline) on an orbital shaker at 37°C for 2 weeks before use. BCG was diluted with 1× Dulbecco’s phosphate-buffered saline (PBS) (Cellgro, Herndon, VA, USA) to 3×10⁸ organisms/mL, the concentration was estimated using McFarland standards (Sigma), and confirmed by plating dilutions onto 7H11 agar plates (Remel, Lenesa, KS, USA). Plates were incubated at 37°C for 3–4 weeks, and resulting colonies counted.

Recombinant mouse and human LFs produced in CHO stable cell lines were provided by Pharma Review Corporation (Houston, TX, USA), manufactured under endotoxin-free, serum-free conditions (Genscript, Piscataway, NJ, USA).^{31 (link),32 (link)} The purity of the CHO-derived LFs was determined by HPLC and SDS PAGE, showing near homogeneity in protein (>98% purity). The methods for purification of the recombinant human LF have been published.^{31 (link)} The level of endotoxin was <10 EU/mg protein. BCG M. bovis, Pasteur strain (TMC 1011; ATCC, Manassas, VA, USA) was grown in Dubos base (without addition of glycerol) with 10% supplement (5% BSA, 7.5% dextrose) on an orbital shaker at 37°C for 2 wk before use. BCG was diluted with 1 × PBS to 3 × 10⁸ bacteria/ml, estimated using McFarland standards (Sigma-Aldrich, St. Louis, MO, USA) and confirmed by plating dilutions onto 7H11 agar plates (Remel, Lenesa, KS, USA). Plates were incubated at 37°C for 3–4 wk, and colonies were enumerated.