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122 lines
4.5 KiB
YAML
122 lines
4.5 KiB
YAML
---
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# Protocol metadata
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id: PROT-0036
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name: Subculturing Cells Protocol
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version: 1.0
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description: Protocol for passaging and subculturing adherent cell lines
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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: Cell culture flask with cells
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notes: 70-90% confluent
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- name: PBS (Phosphate Buffered Saline)
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concentration: 1X, sterile
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temperature: Room temperature
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- name: Tryp-LE or trypsin-EDTA
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temperature: Pre-warmed to 37°C
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storage: -20°C (stock), 4°C (working)
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- name: Complete media
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temperature: Pre-warmed to 37°C
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- name: New culture flask
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type: T25, T75, or T175 as needed
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- name: 70% ethanol
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preparation: Freshly prepared or commercially available
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use: Surface disinfection
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# Equipment required
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equipment:
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- name: Biosafety cabinet
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certification: Class II
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- name: CO2 incubator
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settings: 37°C, 5% CO2, humidified
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- name: Centrifuge
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settings: 200-300g for 5 minutes
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- name: Aspiration system
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type: Vacuum or manual
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- name: Hemocytometer or cell counter (optional)
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use: Cell counting if precise seeding is required
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# Protocol steps
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steps:
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- step: 1
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action: "Prepare workstation"
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details: "Turn on biosafety cabinet, sterilize surfaces and materials with 70% ethanol"
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- step: 2
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action: "Maintain sterile technique"
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details: "Spray gloved hands with 70% ethanol after touching anything outside the hood"
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- step: 3
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action: "Aspirate old media"
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details: "Carefully remove all media without disturbing cell layer"
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- step: 4
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action: "Wash cells with PBS"
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details: "Add sufficient PBS to cover the cell layer, gently rock, then aspirate"
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- step: 5
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action: "Add dissociation reagent"
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details: "Add 1 ml Tryp-LE per T25 flask (scale accordingly for larger flasks)"
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- step: 6
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action: "Incubate cells"
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details: "Place in incubator for 5-10 minutes until cells detach"
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- step: 7
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action: "Check cell detachment"
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details: "Observe under microscope to confirm cells are rounded and detached"
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- step: 8
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action: "Neutralize and collect cells"
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details: "Add 9 ml complete media, pipette gently to dislodge and disperse cells"
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- step: 9
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action: "Seed new flask"
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details: "Transfer 1 ml cell suspension to new flask containing 9 ml complete media (1:10 split ratio)"
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- step: 10
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action: "Label flask"
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details: "Note cell line, passage number, date, and your initials"
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- step: 11
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action: "Incubate cells"
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details: "Place in incubator at 37°C with 5% CO₂"
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# Critical parameters
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critical_parameters:
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- parameter: "Confluence level"
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details: "Ideal passage at 70-90% confluence; overconfluent cells may exhibit altered properties"
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- parameter: "Dissociation time"
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details: "Excessive trypsinization can damage cells; insufficient time leads to poor detachment"
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- parameter: "Split ratio"
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details: "Adjust based on growth rate and experimental timeline (1:3 to 1:20 depending on cell line)"
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# Troubleshooting
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troubleshooting:
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- problem: "Cells not detaching"
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solution: "Extend incubation time with dissociation reagent, tap flask gently, or try fresh trypsin"
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- problem: "Cell clumping"
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solution: "Pipette gently up and down to create single-cell suspension; use cell strainer if needed"
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- problem: "Poor cell recovery/growth"
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solution: "Check media quality, incubator conditions, and avoid over-trypsinization"
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# Safety considerations
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safety:
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ppe: "Lab coat, gloves, and closed-toe shoes required"
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hazards: "Handle biological materials according to biosafety level requirements"
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# Quality control
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quality_control:
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- check: "Cell morphology"
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criteria: "Cells should maintain typical morphology post-passage"
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- check: "Growth rate"
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criteria: "Consistent doubling time compared to historical data for the cell line"
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# References
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references:
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- "Freshney RI. (2016) Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 7th Edition"
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# Notes
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notes: |
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- This protocol describes a 1:10 split ratio
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- Incubation time with Tryp-LE may vary depending on cell line
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- Maintain sterile technique throughout the procedure
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- Consider counting cells for experiments requiring precise cell numbers
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- Record passage number with each subculture
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- Some cell lines may require specialized coating on flasks (collagen, poly-L-lysine, etc.)
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- Check specific requirements for your cell line, as some may need different media or culture conditions
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