The C. Albicans strain used in this study was SC5314. The strain was maintained on solid YED medium and incubated at 30 degrees for at least 2 days.
RAW 264.7 murine macrophages were obtained from the American Type Culture Collection (Rockville, Md) and maintained in complete culture media at 37ºC in a humidified atmosphere with 5% CO2. Cells were maintained at low densities (75% confluence) and passed until reaching the confluent state, usually every 3-4 days on sterile culture plates. RAW cells were plated at the indicated density 18-24 h prior to initiation of the experiments.
The C. Albicans strain used in this study was SC5314. The strain was maintained on solid YED medium and incubated at 30 degrees for at least 2 days.
Extracted molecule
total RNA
Label
Cy3
Hybridization protocol
cDNA was synthesized from 20-30 microgrammes of total RNA by reverse transcription using CyScribe Post-Labeling kit, incorporating Cy3-cUTP or Cy5-dUTP (Amersham Biosciences) into the cDNA corresponding to each sample to be compared. The amount of cDNA as well as the incorporation of Cy3 and Cy5 dyes into cDNA targets was quantified on an Ultrospec 3300 Pro UV/ Visible spectrophotometer (Amersham Biosciences). Both labeled cDNA populations were combined, dried in a vacuum trap, and used as a hybridization probe after resuspension in hybridization solution and 100 micrograms Salmon Sperm. Slides were hybridized overnight with labeled probe at42 degrees in a water bath. Before scanning, the chips were washed and dried following the manufactures instructions
Scan protocol
Microarrays were scanned with a GenePix 4000B scanner (Axon Instruments. Union City, CA) at a resolution of 5m (PMT values ranging from 550 to 700 and laser power 100%)
Description
18-24 h prior to initiation of the co-culture experiment, macrophages were collected and counted with a Neubauer chamber (hemocytometer). A total of 3,5 x 107 cells were plated in complete culture media in 750-ml cell culture flask and grown overnight at 37ºC in a humidified atmosphere with 5% CO2. C. albicans strain SC5314 was grown overnight at 30ºC on solid YED medium (to maintain cells in the yeast form). On day 1, these yeast cells were harvested, washed twice with phosphate buffered saline (PBS), counted and diluted to the desired density in 50 ml of complete culture media. 5 x 107 yeast cells were added per flask to obtain a fungus-macrophage ratio 1:1, (since repeated cell counts of the overnight macrophage cultures indicated a growth rate of 1,4) and incubated for 1.5 h. (for microarray experiments) and 3h. at 37°C and 5% CO2 . Non ingested and unbound Candida cells were removed by washing 3 times with ice-cold PBS. The macrophages and bounded/ingested yeast cells were dislodged by scraping the flask with rubber scrapers in ice-cold water, and pooled by centrifugation for 10 min at 4000 rpm.The resulting cell pellet was resuspended in a 0.25 % Triton X114 solution (50 mM Tris-HCl, 2mM EDTA, pH 7.5), vortexed 3 times and left/chilled on ice in order to eliminate most of the lysated macrophages. After 30 min, the sample was washed 4-6 times with ice-cold MilliQ water, in order to eliminate any trace of the detergent employed.
Data processing
GenePix Pro 4.0 analysis software (Axon Instruments) was used to locate spots in the microarray with the appropriate grid and to obtain the two Cy3/Cy5 image TIFF files. All images were further processed using GenePix 4.0 software according to manufacturer instructions. Flagged spots and spots with an average intensity minus background below the media of the intensity minus background for all the non flagged spots in any channel (Cy3 or Cy5) were not retained for further analysis. Within this group those spots which showed in at least one channel a value of intensity minus background higher than 5 times the media of the background for all spots in the microarray for this channel were recovered.
The median intensity value calculated from total amount of pixels of the spot for channel 1
MEDIAN_CH1_BKD_RAW
The median intensity value calculated from total amount of local background pixels of the spot for channel 1.
MEDIAN_CH2_RAW
The median intensity value calculated from total amount of pixels of the spot for channel 2.
MEDIAN_CH2_BKD_RAW
The median intensity value calculated from total amount of local background pixels of the spot for channel 2.
NEAT_INTENSITY_CH1
Intensity - Background for channel 1
NEAT_INTENSITY_CH2
Intensity - Background for channel 2
FLAGS
Gives information about the data depending on the value of the flag. Flag 100 is good quality spot. Flag 0 is unflagged spot. Flag -50 is low intensity spot which we discard for our analysis. Flag -25 is spot with low reproducibility beteen replicates which we discard for our analysis. Flag -100 is a bad quality spot due to dust, defects in shape, etc which we also discard for our analysis
PRE_VALUE
Ratio of neat intensities for each channel. Ratio is calculated dividing the treatment channel between control channel
