Analysis of Staphylococcus aureus Gene Expression During PMN Phagocytosis - Bacterial Pathogenesis: Methods and Protocols - page 113

6. For each GeneChip, using a clear tube, mix 300 μL of 2× stain buffer, 24 μL
of BSA (50 mg/mL), 6 μL Goat IgG stock (10 mg/mL), 6 μL of biotiny-
lated antibody (0.5 mg/mL), and 264 μL of distilled H 2 O. This is the antibody
solution mix.
7. Remove the GeneChips from the hybridization oven and place a 200-μL pipette
tip into one of the septa. Remove the hybridization cocktail and place into the
appropriate sample tube. These samples can be run on other chips at a later date.
Store unused samples at -80 °C.
8. Fill the GeneChip with 250 μL of wash buffer A without air bubbles ( see

Note 5 ). If processing more GeneChips than allowed in the fluidics station, the
remaining filled chips can be stored up to4hat4°C.
9. In GCOS, click on the “expts” icon and create an experiment (*.EXP) for each
chip.
10. In GCOS, click on “tools,” and then “filters.” Set the filters so your experiments
can be viewed.
11. In GCOS fluidics window, select the appropriate protocol for each module and
run.
12. Place the appropriate GeneChip into the fluidics modules and place the SAPE
solution mix tubes into sample holder positions 1 and 3 for each module.
13. Place the antibody solution mix tube into sample holder position 2 for each
module.
14. After about 90 min, the protocol should be finished. Once the fluidics station
displays “remove cartridge,” take the chip out and inspect it for air bubbles. If
any are present, place back into fluidics module and let it refill. If there are still
bubbles, then the chip must be filled manually with wash buffer A ( see Note 5 ).
15. Place a tough spot over each septum for each GeneChip to be scanned.
16. Gently wipe the glass of the GeneChip with a tissue in one direction. Do not
change direction, as the paraffin at the edges may coat the glass, making scanning
difficult.

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3.10. Scanning
The scanning protocol is written using the 3000 7G (plus) with autoloader.
If an autoloader is not present, the protocol must be altered to accommodate
single-chip scanning. Instruments with serial number starting with 501 or earlier
will require the high-resolution scanning update.

1. Press the start button on the scanner.
2. Open GCOS and assure the connection between the scanner and workstation has
been made.
3. Allow scanner to warm up for 10 min.
4. Place chips into the autoloader starting with position one.