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133 lines
4.7 KiB
YAML
133 lines
4.7 KiB
YAML
---
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# Protocol metadata
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id: PROT-0034
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name: Treating Cells Protocol
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version: 1.0
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description: Protocol for treating cultured cells with control or treatment media
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author: Lab Staff
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created: 2025-05-07
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last_updated: 2025-05-07
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category: cell-culture
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# Materials required
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materials:
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- name: Culture plate
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type: 96-well, 24-well, or 6-well
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preparation: Cells at appropriate confluence for treatment
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- name: Control media
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temperature: Pre-warmed to 37°C
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storage: 4°C
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- name: Treatment media
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temperature: Pre-warmed to 37°C
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storage: As appropriate for treatment compound
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- name: PBS
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temperature: Room temperature or 37°C
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use: Optional for washing (compound-dependent)
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# Equipment required
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equipment:
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- name: Thermal bead bath
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temperature: 37°C
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use: For warming media
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- name: Biosafety cabinet
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certification: Class II
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- name: Aspirator
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type: Vacuum or manual
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- name: Pipettes and sterile tips
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range: Appropriate for plate format
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- name: CO2 incubator
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settings: 37°C, 5% CO2, humidified
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# Protocol steps
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steps:
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- step: 1
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action: "Prepare treatment media"
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details: "Prepare fresh treatment media with the compound of interest at desired concentration"
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- step: 2
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action: "Pre-warm media"
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details: "Place control and treatment media in thermal bead bath at 37°C for at least 15 minutes"
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- step: 3
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action: "Remove plate from incubator"
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details: "Transfer cell culture plate to biosafety cabinet"
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- step: 4
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action: "Aspirate old media"
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details: "Carefully remove all existing media from wells without disrupting cell layer"
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- step: 5
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action: "Optional wash step"
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details: "If needed, gently wash cells with PBS to remove residual media/serum"
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- step: 6
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action: "Add appropriate volume of media"
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details: "Add pre-warmed control or treatment media according to plate format:
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- 150 μL for 96-well plates
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- 1 mL for 24-well plates
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- 2 mL for 6-well plates"
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- step: 7
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action: "Return to incubator"
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details: "Place plate back in incubator for the duration of the treatment period"
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- step: 8
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action: "Document treatment"
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details: "Record treatment start time, concentration, and other relevant details in lab notebook"
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# Volume reference
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volume_reference:
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- plate_type: "96-well"
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media_volume: "100-200 μL (typically 150 μL)"
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- plate_type: "48-well"
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media_volume: "250-500 μL"
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- plate_type: "24-well"
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media_volume: "0.5-1.0 mL"
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- plate_type: "12-well"
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media_volume: "1.0-1.5 mL"
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- plate_type: "6-well"
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media_volume: "1.5-3.0 mL (typically 2 mL)"
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- plate_type: "10 cm dish"
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media_volume: "8-12 mL"
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# Critical parameters
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critical_parameters:
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- parameter: "Media temperature"
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details: "Cold media can shock cells; ensure proper warming to 37°C"
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- parameter: "Treatment timing"
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details: "Maintain consistent timing between wells to ensure comparable exposure times"
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- parameter: "Media volume"
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details: "Insufficient volume can lead to drying; excessive volume may dilute treatment effect"
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# Troubleshooting
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troubleshooting:
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- problem: "Cell death after treatment"
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solution: "Reduce treatment concentration; check solvent toxicity; verify media pH"
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- problem: "No treatment effect"
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solution: "Increase concentration or duration; verify compound stability; check for interference from media components"
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- problem: "Uneven treatment effects"
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details: "Ensure even distribution of treatment; mix gently if needed; check for edge effects"
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# Safety considerations
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safety:
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ppe: "Lab coat, gloves, and eye protection required"
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hazards: "Follow appropriate handling procedures for treatment compounds (check MSDS)"
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waste: "Dispose of treatment media according to institutional guidelines"
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# Quality control
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quality_control:
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- check: "Vehicle control"
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criteria: "Include wells with vehicle-only (solvent) treatment"
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- check: "Positive control"
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criteria: "If available, include wells with known effective compound"
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- check: "Treatment timing"
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criteria: "Record exact timing of treatment addition for accuracy"
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# References
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references:
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- "Cell culture media manufacturer's recommendations"
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- "Primary literature for specific treatments or compounds"
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# Notes
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notes: |
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- Ensure media is warmed to appropriate temperature before adding to cells
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- Media volume should be adjusted according to the plate format
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- Duration of treatment will depend on the specific experiment
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- For long-term treatments (>24 hours), consider refreshing treatment media
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- Some treatments may require serum starvation prior to administration
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- Document lot numbers of treatment compounds for reproducibility
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- For volatile or light-sensitive compounds, take appropriate precautions
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--- |