Biology Reference
In-Depth Information
is necessary to perform traditional growth curves with the particular strain of S.
aureus of interest to your laboratory to calculate number of CFUs/mL.
2. Opsonize S. aureus in 50% normal human serum for 30 min at 37 °C. Following
opsonization, centrifuge bacteria at
5000 × g for 5 min and resuspend at 10 9 /mL
in RPMI/H.
3. Coat the bottom of 12-well cell culture plates with 20% normal human serum.
Incubate at 37 °C for at least 30 min. Wash plates (three times) with sterile DPBS.
Keep plates on ice or at 4 °C until use. Coat enough wells for all samples during
all time points. This includes samples of PMNs only, PMNs + S. aureus , and
S. aureus only.
4. Place the serum-coated 12-well culture plates on ice. To one plate, add 1 mL
of RPMI/H containing 10 7 purified PMNs to each well. To another plate, add
900 μL of RPMI/H.
5. Add 100 μL of 10 9 /mL opsonized S. aureus to all wells except for PMN only
control wells.
6. Centrifuge plates at 4 °C at 500 × g for 8 min to synchronize phagocytosis. Spin
all plates even if wells contain only S. aureus or only PMNs.
7. After centrifugation, incubate plates at 37 °C for desired amounts of time. For
example, a time course may consist of 30, 60, and 180 min.
3.3. Purification of RNA from S. aureus During/After Phagocytosis
by Human PMNs
1. At desired time points, harvest samples by removing all liquid from the wells
(
1.1 mL) and centrifuging at 5000 × g for 5 min (at room temperature).
Immediately after removing all supernatant, add 700 μL of RLT lysis buffer
(containing 143 m M -ME) to the wells ( see Note 1 ). Swirl lysis buffer to
completely coat the bottom of the wells. Pipet lysate to ensure complete lysis
of the adherent portion of the sample. When centrifugation is finished, decant
supernatant and lyse pellet with the lysis buffer used to lyse the adherent cells
in the corresponding well. Vigorously pipet and vortex the sample.
2. Add each sample to a 2-mL tube of Lysing Matrix B. Physically disrupt sample
using a FastPrep instrument at 6 m/s for 20 s.
3. Centrifuge sample (in the Lysing Matrix B tube) at 600 × g for 4 min (this
centrifugation is simply to “settle” the silica spheres).
4. Pipet sample into new 1.7-mL microcentrifuge tubes. Approximately 500 μL of
lysate will be recovered.
5. Add 350 μL of ethanol to the sample. Mix sample by pipetting.
6. Apply 700 μL of the sample (including any precipitate) to an RNeasy mini
column placed in a 2-mL collection tube. Centrifuge sample for 60 s at
8000
× g and discard the flow-through. Run any excess sample through the same
column as described.
7. Wash the column with 700 μL of buffer RW1 (from RNeasy kit) and centrifuge
sample for 60 s at
8000 × g . Discard the flow-through.
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