3 Material and Methods
3.2 Methods
3.2.1 Depletion of specific IgGs from IVIG
S. aureus ATCC 29213 or E coli K12 XL1blue from an overnight (ON) culture was inocu-
lated 1:100 in 400 ml LB and cultured until an optical density of 2.0 at 600 nm (OD
600
) was
reached. Subsequently bacteria were harvested by centrifugation, washed in phosphate-
buffered saline (PBS) (pH 7.3) and resuspended in an IVIG preparation (Octagam
®
, Oc-
tapharma) or PBS as control. The suspension was rotated slowly ON at 4 ° C, subsequently
bacteria were removed by centrifugation, and the supernatant was sterile filtered through a
0.2 µm membrane filter. Protein concentration of IVIG depleted of S. aureus (dSaIVIG) or
E. coli specific IgGs (dEcIVIG) was determined using the Bio-RAD DC assay according to
the manufacturer’s instructions.
3.2.2 S. aureus growth curves
S. aureus ATCC 29213 from ON culture was inoculated 1:100 into LB broth and cultured
until OD
600
of 0.3 was reached. Subsequently bacteria were harvested by centrifugation at
4 ° C, 3000 g for 15 minutes. After washing once with cold PBS, the number of S. aureus
colony forming units (CFU) was estimated by OD measurement, based on the observation
that an OD
600
of 3 equates to 1 x 10
9
CFU/ml. For experimental assays 1 x 10
4
CFU were
inoculated in 500 µl LB broth. 13 mg / ml of either IVIG, BSA, dialysed IVIG (IVIG-DS),
dSaIVIG or dEcIVIG adjusted in 500 µl PBS or solely PBS were added (final volume =
1 ml). Where indicated 25 or 50 % of the total volume (1 ml) were substituted by human
serum, heat-inactivated (HI) human serum, IVIG or PBS (control). At defined time points
(every hour up to 5 hours) samples were taken, diluted 1:2 in 0.1 % Triton X 100, sonicated
for 5 minutes and 50 µl of suitable dilutions were plated on Mueller Hinton agar using the
Eddy jet spiral plater (IUL Instruments). Plates were incubated at 37 ° C for 16 hours and
CFUs were enumerated using the Countermat Flash (IUL Instruments). Experiments were
performed using triplicates.
To enable expression profiling during the course of growth inhibition, RNA was isolated
from S. aureus cultured in the presence of IVIG, dSaIVIG or PBS. In a total volume of 10 ml,
1 x 10
7
CFU / ml were co-incubated with either 2.5 mg / ml IVIG, dSaIVIG or PBS substituted
with 5 mg / ml Maltose (adjusted to the concentration present in IVIG) in a 1:1 ratio with LB
broth. To assess growth of S. aureus under these conditions, triplicate samples for each
treatment were included in parallel to the samples for RNA isolation. At defined time points
(every 30 minutes up to 120 minutes, or immediately before RNA isolation) a sample was
taken, diluted 1:2 in 0.1 % Triton X 100, sonicated for 5 minutes and 50 µl of suitable dilutions
were spirally plated on Mueller Hinton agar. Plates were incubated at OD 37 ° C for 16 hours
and CFUs were enumerated. RNA samples were immediately harvested by centrifugation
23
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