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# Protocol metadata
id: PROT-0036
name: Adipogenic Induction Treatment
version: 1.0
description: Protocol for inducing adipogenesis in preadipocyte cells using a combination of IBMX, dexamethasone, and insulin
author: James M. Jordan
created: 2025-05-07
last_updated: 2025-05-07
category: cell-treatment

# Materials required
materials:
  - name: 3-Isobutyl-1-methylxanthine (IBMX)
    concentration: 0.5 mM final
    storage: -20°C
    preparation: Dissolve in DMSO to make 500X stock (250 mM)
    supplier: Sigma-Aldrich (I5879)
  - name: Dexamethasone
    concentration: 1 µM final
    storage: -20°C
    preparation: Dissolve in ethanol to make 1000X stock (1 mM)
    supplier: Sigma-Aldrich (D4902)
  - name: Insulin
    concentration: 10 µg/mL final
    storage: -20°C
    preparation: Dissolve in acidified water (pH 4.5) to make 1000X stock (10 mg/mL)
    supplier: Sigma-Aldrich (I6634)
  - name: DMEM high glucose
    storage: 4°C
    supplier: Gibco
  - name: Fetal Bovine Serum (FBS)
    concentration: 10% final
    storage: -20°C (aliquots)
    supplier: Gibco
  - name: Penicillin-Streptomycin
    concentration: 1% final
    storage: -20°C
    supplier: Gibco
  - name: Complete growth medium
    composition: DMEM + 10% FBS + 1% Pen-Strep
    storage: 4°C

# Equipment required
equipment:
  - name: Biosafety cabinet
    certification: Class II
  - name: CO2 incubator
    settings: 37°C, 5% CO2, humidified
  - name: Water bath
    settings: 37°C
  - name: Serological pipettes
    sizes: 5 mL, 10 mL, 25 mL
  - name: Micropipettes
    sizes: P1000, P200, P20

# Protocol steps
steps:
  - step: 1
    action: "Prepare complete growth medium"
    details: "To 500 mL DMEM high glucose, add 50 mL FBS and 5 mL Pen-Strep. Mix well and warm to 37°C before use."
  - step: 2
    action: "Thaw induction reagent stocks"
    details: "Remove IBMX, dexamethasone, and insulin stock solutions from -20°C and thaw at room temperature. Protect from light."
  - step: 3
    action: "Prepare adipogenic induction medium (AIM)"
    details: "To complete growth medium, add IBMX (final 0.5 mM), dexamethasone (final 1 µM), and insulin (final 10 µg/mL). Mix thoroughly but gently by inverting."
  - step: 4
    action: "Warm media"
    details: "Warm both complete growth medium (control) and adipogenic induction medium to 37°C before adding to cells."
  - step: 5
    action: "Aspirate existing medium from cells"
    details: "Using a sterile aspirator, carefully remove all existing medium from the cell culture vessel."
  - step: 6
    action: "Add fresh medium"
    details: "Add appropriate volume of either complete growth medium (control) or adipogenic induction medium to the cells."
  - step: 7
    action: "Return cells to incubator"
    details: "Place cell culture vessels in 37°C, 5% CO2 incubator."
  - step: 8
    action: "Maintain treatment"
    details: "For standard protocol, maintain cells in adipogenic induction medium for 3 days, then switch to insulin-only medium (10 µg/mL insulin in complete medium) for additional 4-11 days."

# Critical parameters
critical_parameters:
  - parameter: "Cell confluence"
    details: "Cells should be at 100% confluence at the time of induction. Post-confluent cells (2 days after reaching confluence) often yield better differentiation."
  - parameter: "Reagent concentration"
    details: "IBMX (0.5 mM), dexamethasone (1 µM), and insulin (10 µg/mL) concentrations are critical. Prepare fresh stocks if uncertain about stability."
  - parameter: "Media change frequency"
    details: "After the initial 3-day induction period, change to insulin-only medium and then change medium every 2-3 days for optimal differentiation."

# Troubleshooting
troubleshooting:
  - problem: "Poor differentiation"
    solution: "Ensure cells were 100% confluent before induction; check reagent quality and concentrations; extend post-confluent period to 2 days before induction."
  - problem: "Cell detachment"
    solution: "Handle cells gently during media changes; ensure plate surface is appropriate for adipocyte culture; consider using collagen-coated plates."
  - problem: "Contamination"
    solution: "Use sterile technique; check medium and reagents for contamination; consider adding additional antibiotics."

# Safety considerations
safety:
  ppe: "Lab coat, gloves, and eye protection required"
  hazards: "DMSO (IBMX solvent) can enhance skin penetration of other chemicals; dexamethasone is a synthetic glucocorticoid with potential health effects."
  disposal: "Dispose of media and solutions according to institutional guidelines for biological waste."

# Expected outcomes
expected_outcomes:
  - outcome: "3T3-L1 cells should begin showing lipid droplet formation within 3-5 days"
  - outcome: "Maximum differentiation typically reached by day 8-10"
  - outcome: "Adipogenic marker genes (PPARγ, C/EBPα, FABP4, etc.) upregulated within 1-2 days"
  - outcome: "Early adipogenic transcription factors (C/EBPβ, C/EBPδ) upregulated within hours"

# References
references:
  - "Zebisch K, et al. (2012) Protocol for effective differentiation of 3T3-L1 cells to adipocytes. Anal Biochem. 425(1):88-90."
  - "Green H, Kehinde O. (1975) An established preadipose cell line and its differentiation in culture II. Factors affecting the adipose conversion. Cell. 5(1):19-27."
  - "Rubin CS, et al. (1978) Development of hormone receptors and hormonal responsiveness in vitro. Insulin receptors and insulin sensitivity in the preadipocyte and adipocyte forms of 3T3-L1 cells. J Biol Chem. 253(20):7570-7578."

# Notes
notes: |
  - This protocol is optimized for 3T3-L1 cells but can be adapted for other preadipocyte cell lines or primary cells.
  - Cell response to adipogenic induction can vary between passages, so consistency in culture conditions is important.
  - For experiment termination at 24h post-induction, cells will only show early adipogenic markers (C/EBPβ, C/EBPδ) but not mature adipocyte phenotype.
  - YBX1 has been reported to interact with C/EBPα during early adipogenesis as part of transcriptional regulation.
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