Project protocol — Contents
Workflow and sampling
Equipment
Reagents, supplies, and solutions
Procedure
Definitions and calculations
Data
References
Workflow
200 µL 5 x 104 blastospores
Kidneys harvested and processed 48h following infection Kidney homogenates cultured and assessed for fungal load Complement component 5 (C5) allele status determined (Hc gene)
- Hemocytometer
- Cell culture incubator heated at 30°C
- Small rodent dissecting kit
- Tissue homogenizer
- Centrifuge
- Cell/colony counter
- YPD medium (1% yeast extract, 2% Bacto Peptone and 2% dextrose)
- Phosphate-buffered saline (PBS)
- YPD-agar plates containing 34 mg/mL of chloramphenicol
- 3 mL syringes
- 19 gauge needles
- Cell culture plates
- Cell culture tubes
I. Infection with Candida albicans and assessment of kidney fungal load
a. C. albicans strain SC5314 is grown overnight in YPD medium at 30°C.
b. Blastospores are harvested by centrifugation, washed twice in PBS, counted using a hemacytometer and resuspended in PBS at the required density.
c. Female mice are inoculated via the tail vain with 200 µL of a suspension containing 5 x 104 C. albicans blastospores in PBS.
d. Kidneys are aseptically removed 48h after infection and homogenized in 5 mL of PBS.
e. Kidney homogenate is serially diluted and plated on YPD-agar plates containing 34 mg/mL of chloramphenicol.
f. Plates are incubated at 30°C for 24â48 h and the colony-forming units (CFU) counted and expressed as log10CFU per kidney (maximum sensitivity for this assay is 100 CFU; mice displaying titers below the detection limit are assigned an arbitrary value of 100 CFU).
g. Strains are designated susceptible (=0) or resistant (=1) based on CFU strain means, where susceptible is assigned when log10CFU per kidney > 5.1; resistant is assigned when log10CFU per kidney < 4.2 (Note: designations for susceptible (=0) or resistant (=1) for each strain are assigned to individual animals in the dataset).
II. Evaluating C5 status
Strains are determined to be C5-sufficient or C5-deficient (loss of function): C5 allele status is determined by genotyping genomic tail DNA obtained from JAX® (Note: C5 allele status was determined on one DNA sample for each strain; genotype information is provided for individual mice in the dataset although individual mice in the study were not genotyped). For more information about the C5 deletion, see Wetsel et al.; for information about C5 primers and other procedural details, see Tuite et al.
Submitting investigator's notes: "Despite being C5-sufficient and producing serum C5a during infection (data not shown), SM/J mice display a significantly higher mean fungal load (log10CFU = 5.2 ± 0.4) when compared to the reference strain, C57BL/6J (log10CFU = 4.1 ± 0.5), or other C5-sufficient strains. AKR/J mice are C5-deficient, yet they showed mean log10CFU counts at 3.9 ± 0.4 similar to that seen in the resistant C57BL/6J strain, and clearly distinct from that seen in the C5-deficient group (log10CFU>5.1)."
Data collected by investigator
• fungal load in the kidney
• susceptibility to candidiasis
• complement component 5 (C5) allele status
Candidiasis: mycotic or fungal infections of yeasts (i.e. Candida albicans) origin.
Complement component 5 (C5): is a protein component of the complement system, which when activated results in complement cascade critical for pathogen clearance.
Colony forming units (CFU): is a measure of viable bacterial or fungal numbers.
C. albicans susceptible group = log10CFU > 5.1 (per kidney)
C. albicans resistant group = log10CFU < 4.2 (per kidney)
References
Wetsel RA, Fleischer DT, Haviland DL. Deficiency of the murine fifth complement component (C5). A 2-base pair gene deletion in a 5'-exon. J Biol Chem. 1990 Feb 15;265(5):2435-40.
PubMed 2303408