Source Paper
Materials & Equipment Checklist
8 items2 from ConductScience
Gather these items before starting the experiment. Check off items as you prepare.
Equipment3
Materials4
Jackson Labs • not applicable • not specified • not specified
Indiana University School of Medicine (breeding pairs donated by Dr. Xiao-Ming Xu) • not applicable • not specified • not specified
Jackson Labs • not applicable • not specified • not specified
not specified • not specified • not specified • not specified
Software1
NanoString Technologies • not specified
2 items available from ConductScience
Estimated: $12,510.00
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Protocol Steps
1
Animal acquisition and breeding
Obtain C57BL/6 mice (3 months old) from Jackson Labs as HSCT recipients. Obtain cPLA2 KO breeding pairs from Indiana University School of Medicine and backcross to C57BL/6J females from Jackson Labs. Obtain Actin-GFP mice from Jackson Labs.
not specifiednot specified
Note: All animals housed in IVC cages with ad libitum food and water access
View evidence from paper
“Three-month-old C57BL/6 mice (Jackson Labs, Bar Harbor, Maine) were used as HSCT recipients. cPLA2 KO mice were backcrossed to C57BL/6J females acquired from Jackson Labs.”
2
Hematopoietic stem cell transplantation (HSCT)
Perform HSCT using donor cells from cPLA2 KO, cPLA2 WT, or Actin-GFP mice into C57BL/6 recipient mice
not specifiednot specified
Note: Backcrossing to C57BL/6J reduces risk of bone marrow rejection and graft versus host disease
View evidence from paper
“To reduce the risk of bone marrow rejection or related pathologies (graft versus host disease), cPLA2 KO mice were backcrossed to C57BL/6J females”
3
T9 spinal cord injury induction
Induce T9 spinal cord injury in female mice at 8 weeks post-HSCT (Cohort B) or 7 days post-HSCT (Cohort C)
not specifiednot specified
Note: Cohort B: WT→WT n=16, cPLA2KO→WT n=16 pre-exclusion. Cohort C: WT→WT n=6, cPLA2KO→WT n=6
View evidence from paper
“All animals T9 SCI WT→WT=16 cPLA2KO→WT=16 post-exclusion WT→WT=14 cPLA2KO→WT=13”
4
Behavioral assessment - BMS scoring
Assess locomotor recovery using BMS (Basso Mouse Scale) scoring at specified timepoints
6 weeks post-SCI for Cohort Bnot specified
Note: Exclusion criteria: BMS score ≥3 at 1 dpi (days post-injury)
View evidence from paper
“Behavioral analysis of locomotor recovery: BMS, Horizontal Ladder, Catwalk gait analysis. 2 mice from WT group and 1 mouse excluded from cPLA2 KO group for meeting a priori exclusion criteria (BMS score ≥ 3 at 1 dpi)”
5
Behavioral assessment - Horizontal Ladder test
Assess locomotor recovery and coordination using Horizontal Ladder test
6 weeks post-SCI for Cohort Bnot specified
Note: Part of behavioral analysis battery
View evidence from paper
“Behavioral analysis of locomotor recovery: BMS, Horizontal Ladder, Catwalk gait analysis”
6
Behavioral assessment - Catwalk gait analysis
Analyze gait and locomotor recovery using Catwalk gait analysis system
6 weeks post-SCI for Cohort Bnot specified
Note: Part of behavioral analysis battery
View evidence from paper
“Behavioral analysis of locomotor recovery: BMS, Horizontal Ladder, Catwalk gait analysis”
7
Tissue collection and pathology analysis
Collect spinal cord tissue at 6 weeks post-SCI (Cohort B) for assessment of spared tissue and intralesional axons
6 weeks post-SCInot specified
Note: Tissue pathology outcomes measured
View evidence from paper
“Tissue pathology: Spared tissue and intralesional axons 6 weeks”
8
Gene expression analysis
Analyze gene expression of spinal cord homogenate using nCounter gene array (NanoString Technologies) at 7 days post-SCI (Cohort C)
7 days post-SCInot specified
Note: Cohort C endpoint
View evidence from paper
“Gene expression of spinal cord homogenate by nCounter (NanoString Technologies) gene array 7 days”
9
Leukocyte cPLA2 expression assessment
Measure leukocyte cPLA2 expression at endpoint for exclusion criteria
at 6 weeks post-SCInot specified
Note: 2 mice from cPLA2 KO group excluded based on elevated leukocyte cPLA2 expression
View evidence from paper
“2 mice from cPLA2 KO group excluded based on elevated leukocyte cPLA2 expression at endpoint.”
Subjects / Specimens
- Species
- mouse
- Strain
- C57BL/6, cPLA2 KO, cPLA2 WT littermate controls, Actin-GFP C57BL/6
- Age
- 2-4 months (donors), 3 months (HSCT recipients)
- Sex
- female
- Count
- 29
- Weight
- not specified
C57BL/6 mice obtained from Jackson Labs, Bar Harbor, Maine. cPLA2 KO mice backcrossed to C57BL/6J females from Jackson Labs. Actin-GFP mice originally from Jackson Labs.