docs/protocols/preparation_of_complete_dme_f12_itsd_v1.yaml

---
# Protocol metadata
id: PROT-0032
name: Preparation of Complete DME/F-12 ITSD Protocol
version: 1.0
description: Protocol for preparing complete DME/F-12 with ITS and dexamethasone supplements
author: Lab Staff
created: 2025-05-07
last_updated: 2025-05-07
category: reagent-preparation

# Materials required
materials:
  - name: DME/F-12 medium
    amount: 500 mL bottle
    storage: 4°C
    supplier: Recommended supplier
  - name: Fetal Bovine Serum (FBS)
    amount: 50 mL (10% final)
    storage: -20°C (stock), thawed before use
    supplier: Recommended supplier, heat-inactivated
  - name: Insulin-Transferrin-Selenium (ITS)
    amount: 5 mL (1% final)
    storage: -20°C
    supplier: Commercial 100X solution
  - name: Dexamethasone
    amount: 5 μL of 10 mM stock (1 nM final)
    storage: -20°C
    preparation: Stock in ethanol or DMSO
  - name: Penicillin-Streptomycin (PS)
    amount: 5 mL (1% final)
    storage: -20°C
    supplier: Recommended supplier, 100X concentration
  - name: Sterile 50-mL tube
    type: Conical, polypropylene
    preparation: Sterile

# Equipment required
equipment:
  - name: Biosafety cabinet
    certification: Class II
  - name: Water bath or incubator
    temperature: 37°C
    use: For thawing frozen supplements
  - name: Pipette aid
    range: For 5-50 mL volumes
  - name: Micropipettes
    range: For small volumes (dexamethasone)
  - name: Serological pipettes
    range: Various sizes
  - name: Refrigerator
    temperature: 4°C
    use: For media storage

# Protocol steps
steps:
  - step: 1
    action: "Prepare materials"
    details: "Allow media to warm to room temperature; thaw FBS, ITS, and PS at 37°C"
  - step: 2
    action: "Set up biosafety cabinet"
    details: "Turn on biosafety cabinet 15 minutes before use; clean with 70% ethanol"
  - step: 3
    action: "Aliquot plain media"
    details: "Transfer ~55 mL of DME/F-12 media into a sterile 50-mL tube and label as 'PLAIN DME/F-12'"
  - step: 4
    action: "Add FBS"
    details: "To the remaining 445 mL of media in the original bottle, add 50 mL Fetal Bovine Serum (FBS)"
  - step: 5
    action: "Add ITS supplement"
    details: "Add 5 mL Insulin-Transferrin-Selenium (ITS) solution to the main bottle"
  - step: 6
    action: "Add dexamethasone"
    details: "Add 5 μL of 10 mM dexamethasone stock solution (final concentration 1 nM)"
  - step: 7
    action: "Add antibiotics"
    details: "Add 5 mL Penicillin-Streptomycin (PS) solution to the main bottle"
  - step: 8
    action: "Mix gently"
    details: "Invert bottle several times to mix thoroughly, avoiding bubble formation"
  - step: 9
    action: "Label media"
    details: "Label bottle with 'Complete DME/F-12 ITS-D', date of preparation, and expiration date (2-3 weeks from preparation)"
  - step: 10
    action: "Store media"
    details: "Store both complete and plain media at 4°C; protect from light if possible"

# Final composition
final_composition:
  - component: "DME/F-12 basal medium"
    percentage: "88%"
    function: "Provides essential nutrients and buffering"
  - component: "Fetal Bovine Serum"
    percentage: "10%"
    function: "Provides growth factors and proteins for cell growth"
  - component: "Insulin-Transferrin-Selenium"
    percentage: "1%"
    function: "Insulin: promotes glucose uptake; Transferrin: iron carrier; Selenium: antioxidant"
  - component: "Dexamethasone"
    concentration: "1 nM"
    function: "Glucocorticoid that promotes cell differentiation in certain cell types"
  - component: "Penicillin-Streptomycin"
    percentage: "1%"
    function: "Prevents bacterial contamination"

# Critical parameters
critical_parameters:
  - parameter: "Sterility"
    details: "All components must be handled aseptically in a biosafety cabinet"
  - parameter: "Dexamethasone concentration"
    details: "Accurate pipetting of small volume (5 μL) is critical; consider using serial dilutions"
  - parameter: "Expiration"
    details: "Media with ITS may have shorter shelf life (2-3 weeks) compared to standard media"

# Troubleshooting
troubleshooting:
  - problem: "Precipitate formation"
    solution: "May occur with ITS; warm to room temperature and mix gently; filtration may be required"
  - problem: "Cell differentiation issues"
    solution: "Verify dexamethasone concentration; prepare fresh medium; check ITS potency"
  - problem: "Media color change"
    solution: "If phenol red indicator turns yellow, pH is too acidic; discard media"

# Safety considerations
safety:
  ppe: "Lab coat, gloves required"
  hazards: "Minimal hazards; follow standard laboratory safety practices"
  dexamethasone: "Potent steroid hormone; avoid skin contact"

# Quality control
quality_control:
  - check: "pH indicator"
    criteria: "Phenol red should appear pink-red (not yellow or purple)"
  - check: "Sterility test"
    criteria: "Incubate a sample at 37°C for 72 hours to verify no contamination"
  - check: "Functional test"
    criteria: "Test with appropriate cell differentiation model if possible"

# References
references:
  - "ATCC. Guidelines for Media Preparation and Methods. www.atcc.org"
  - "Freshney, R.I. Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 7th Edition"
  - "Barnes D, Sato G. (1980) Methods for growth of cultured cells in serum-free medium. Analytical Biochemistry 102(2):255-270"

# Notes
notes: |
  - This specialized medium contains additional supplements:
    - 10% FBS
    - 1% ITS
    - 1 nM dexamethasone
    - 1% Penicillin-Streptomycin
  - ITS supplement is typically used to reduce serum requirements or enhance differentiation
  - Dexamethasone concentration (1 nM) is suitable for differentiation of certain cell types
  - For more potent glucocorticoid effects, concentration can be increased to 10-100 nM
  - For long-term studies, consider preparing smaller volumes of media more frequently
  - Some cell types may require adaptation period when switching to ITS-supplemented media
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