For competitive repopulation assays, B220 and CD3-depleted bone marrow cells from donor CD45.1 control mice were mixed with the same type of cells derived from either CD45.2 control or CD45.2 PDK1fl/fl/Vav-Cre+ve donor mice in 1 1 ratio.
As a further check for high-fidelity sorting, donor cells (GFP- or GFP+) were mixed with genetically tagged (GFP+ or GFP-) cells (Additional file 9C) and the genotype of the resultant outgrowths was checked by immunohistochemistry for GFP (Additional file 9D).
Total IgG (containing TBV induced antibody) purified from vaccinated mice were mixed with the donor blood samples.
For conjugation, 4 recipients of B. henselae EryR resuspended in brain heart infusion broth (BHI) were mixed with one donor of B. rattaustraliani (AUST/NH4T), the mixture was incubated at 37°C for 2 hours to overnight without shaking.
Chondrocytes were cultured in 96-well plates (5 × 10) and stimulated with different NO donors for 24 hours; 10 μl of supernatant were mixed with 10 μl of lactate reagent and incubated for 5 minutes at room temperature.
Approximately 150 ng of PCR fragment and a donor vector (pDONR221 pDONRr, pDONR 221 pDONR, pDONR 221 pDONR3r, pDONR 221 P3-P2) were mixed with 2 µl of BP clonase™ II enzyme mix, and adjusted to 10 µl with TE buffer.
The protocol was modified as follows: Overnight cultures of the donor strain (20 μl) and recipient strain (60 μl) were mixed with 2 mL of fresh Luria-Bertani broth and incubated for 4 h at 37°C.
(iv), Green fluorescence transfer was not detected in recipient cells that were not in contact with donor cells, or in a negative control experiment, when they were mixed with a traA mutant.
Bacteria were mixed with 80 ml of whole blood from each donor (final GBS concentration of ∼5×108 CFU/ml of blood) and incubated at 37°C with slight rotation to avoid sedimentation of blood and bacterial cells.
For whole blood killing assays, inocula of 100 CFU in 100 μL were mixed with 300 μL human blood (n = 5 donors) in heparinized tubes and incubated for 1 3 h with mild agitation.
