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MB Sample ID: SA235724
| Local Sample ID: | RS_M6-1_1 |
| Subject ID: | SU002439 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Genotype Strain: | C57BL/6J |
| Age Or Age Range: | 6-8 weeks |
| Gender: | Female |
| Animal Animal Supplier: | The Jackson Laboratory, Bar Harbor, ME |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002439 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Genotype Strain: | C57BL/6J |
| Age Or Age Range: | 6-8 weeks |
| Gender: | Female |
| Animal Animal Supplier: | The Jackson Laboratory, Bar Harbor, ME |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| RS_M6-1_1 | SA235724 | FL029256 | C. immitis | Subject_ID |
| RS_M6-1_1 | SA235724 | FL029256 | Fungi | treatment |
Collection:
| Collection ID: | CO002432 |
| Collection Summary: | Female C57BL/6J mice (The Jackson Laboratory, Bar Harbor, ME) 6-8 weeks of age were used for these studies. Mice were housed according to NIH guidelines for housing and care in a biosafety level 3 animal laboratory. All procedures were approved by the Institutional Animal Care and Use Committee (protocol number 16-011) of Northern Arizona University. The Coccidioides isolates used in this study were the type strains C. immitis strain RS (ATCC® catalog no. NR-48942; NCBI accession no. AAEC00000000.3) and C. posadasii strain Silveira (ATCC® catalog no. NR-48944; NCBI accession no. ABAI00000000.2). Mice were anesthetized with ketamine/xylene (80/8 mg/kg) and intranasally inoculated with 100 arthroconidia of C. immitis strain RS (n=6) or C. posadasii strain Silveira (n=6) suspended in 30 μL phosphate-buffered saline (PBS), as described previously (22, 58). Control mice were inoculated with PBS alone (n=4). The mice were sacrificed at day 10 post-inoculation. The lungs were rinsed with 2 mL of PBS to collect bronchoalveolar lavage fluid (BALF), which were filtered with 0.22 μm Ultrafree® - MC centrifugal filter devices with Durapore® membrane (MilliporeSigma, Burlington, MA). One milliliter of each BALF sample was stored at –80°C for volatilomics analysis. Halt™ Protease Inhibitor Cocktail (10 μL/mL) was added to the remainder of each BALF sample for cytokine analysis. Spleen and brain were homogenized in 1 ml of sterile PBS followed by culture of 10-fold dilutions of each tissue on 2X GYE agar (2% glucose (VWR™, USA), 1% yeast extract (BD™, Franklin Lakes, New Jersey, USA, and 1.5% bacteriological agar (Difco, USA)) to assess fungal dissemination. |
| Sample Type: | Bronchoalveolar lavage |
Treatment:
| Treatment ID: | TR002451 |
| Treatment Summary: | Mice were anesthetized with ketamine/xylene (80/8 mg/kg) and intranasally inoculated with 100 arthroconidia of C. immitis strain RS (n=6) or C. posadasii strain Silveira (n=6) suspended in 30 μL phosphate-buffered saline (PBS), as described previously (22, 58). Control mice were inoculated with PBS alone (n=4). The mice were sacrificed at day 10 post-inoculation. The lungs were rinsed with 2 mL of PBS to collect bronchoalveolar lavage fluid (BALF), which were filtered with 0.22 μm Ultrafree® - MC centrifugal filter devices with Durapore® membrane (MilliporeSigma, Burlington, MA). One milliliter of each BALF sample was stored at –80°C for volatilomics analysis. Halt™ Protease Inhibitor Cocktail (10 μL/mL) was added to the remainder of each BALF sample for cytokine analysis. Spleen and brain were homogenized in 1 ml of sterile PBS followed by culture of 10-fold dilutions of each tissue on 2X GYE agar (2% glucose (VWR™, USA), 1% yeast extract (BD™, Franklin Lakes, New Jersey, USA, and 1.5% bacteriological agar (Difco, USA)) to assess fungal dissemination. |
Sample Preparation:
| Sampleprep ID: | SP002445 |
| Sampleprep Summary: | The BALF samples were allowed to thaw at 4°C overnight, and then split into technical triplicates of 200 μL that were transferred and sealed into sterilized 2mL GC headspace vials with Supelco® PTFE/silicone septum magnetic screw caps (Sigma-Aldrich®, St. Louis, MO). Samples were randomized for analysis. Volatile metabolites sampling was performed by solid phase microextraction (SPME) using a Gerstel® MPS Robotic Pro MultiPurpose autosampler directed by Maestro® software (Gerstel®, Inc., Linthicum, MD). Sample extraction and injection parameters are provided in Table S3 (see Autosampler Method). Volatile metabolite analysis was performed by two-dimensional gas chromatography−time-of-flight mass spectrometry (GC×GC–TOFMS) using a LECO® Pegasus® 4D and Agilent® 7890B GC (LECO® Corp., St. Joseph, MI). Chromatographic, mass spectrometric, and peak detection parameters are provided in Table S3 (see GC×GC Method and Mass Spectrometry Method). An external alkane standards mixture (C8 – C20; Sigma-Aldrich®) was sampled multiple times for calculating retention indices (RI). The injection, chromatographic, and mass spectrometric methods for analyzing the alkane standards were the same as for the samples. |
| Extraction Method: | Solid-phase microextraction (SPME) |
Combined analysis:
| Analysis ID | AN003836 |
|---|---|
| Analysis type | MS |
| Chromatography type | GC |
| Chromatography system | Agilent 7890B |
| Column | Column 1: Rxi-624Sil MS,(60m × 0.25mm × 1.4um); 2: Stabilwax,(1m × 0.25mm × 0.5um) |
| MS Type | EI |
| MS instrument type | GC x GC-TOF |
| MS instrument name | Leco Pegasus 4D GCxGC TOF |
| Ion Mode | POSITIVE |
| Units | Peak areas |
Chromatography:
| Chromatography ID: | CH002840 |
| Instrument Name: | Agilent 7890B |
| Column Name: | Column 1: Rxi-624Sil MS,(60m × 0.25mm × 1.4um); 2: Stabilwax,(1m × 0.25mm × 0.5um) |
| Chromatography Type: | GC |
MS:
| MS ID: | MS003578 |
| Analysis ID: | AN003836 |
| Instrument Name: | Leco Pegasus 4D GCxGC TOF |
| Instrument Type: | GC x GC-TOF |
| MS Type: | EI |
| MS Comments: | See attached protocol |
| Ion Mode: | POSITIVE |
