Home โ€บ Cell Biology โ€บ How to Passage Cells: Cell Culture Basics Training
Steps
  1. 1 Introduction: Cell Growth and Culture Phases --:--
  2. 2 Preparing the Hood and Supplies 00:40
  3. 3 Removing Medium and Washing Cells 01:30
  4. 4 Adding Dissociation Reagents (TrypLE Express or Trypsin) 02:10
  5. 5 Confirming Cell Detachment with a Microscope 03:00
  6. 6 Centrifugation and Resuspension 04:00
  7. 7 Cell Counting with Trypan Blue 05:00
Cell Biology Thermo Fisher Scientific

How to Passage Cells: Cell Culture Basics Training

Protocol

Cell culture basics: passaging adherent mammalian cells. Covers media check, trypsinization, neutralization, cell counting, and seeding into new flasks.

Difficulty
beginner
Total time
30-45 min
Biosafety
BSL-1

Steps

1
Introduction: Cell Growth and Culture Phases

Overview of the cell growth curve (lag, log, plateau) and why timely passaging matters to keep cells in healthy exponential phase.

โ–ถ --:--
2
Preparing the Hood and Supplies

Set up the biosafety cabinet: spray and wipe with 70% ethanol, organize pre-warmed media, PBS, dissociation reagent, pipettes, and labeled flasks.

โ–ถ 00:40
3
Removing Medium and Washing Cells

Aspirate the old culture medium completely; rinse cells gently with sterile PBS to remove residual serum that would inhibit the dissociation reagent.

โ–ถ 01:30
4
Adding Dissociation Reagents (TrypLE Express or Trypsin)

Add pre-warmed TrypLE Express or trypsin-EDTA, cover the entire monolayer, then incubate at 37 C to detach cells from the surface.

โ–ถ 02:10
5
Confirming Cell Detachment with a Microscope

After 2-5 min, check under the microscope: cells should be rounded up and floating. Tap the flask gently if needed to dislodge stragglers.

โ–ถ 03:00
6
Centrifugation and Resuspension

Neutralize the dissociation reagent with complete media, transfer to a tube, spin at 200-300g for 5 min, discard supernatant, resuspend the cell pellet in fresh media.

โ–ถ 04:00
7
Cell Counting with Trypan Blue

Mix cell suspension with trypan blue (1:1); load into a hemocytometer or automated cell counter; calculate cell density and viability before seeding at desired ratio.

โ–ถ 05:00

๐Ÿšจ Failure Case Library (6) + Submit your own case

severe
Trace Metal Precipitation in Serum-Free Formulations
Copper, iron, and zinc supplements precipitate out of serum-free media, creating a toxic environment for cells. Metal precipitates appear as dark particles and can compromise cell viability due to altered bioavailability and oxidative stress.
๐Ÿ’ก 4 ยท โœ“ 4
severe
Nutrient Loss via Chelation by Precipitates
Cell health deteriorates due to precipitate-mediated removal of essential nutrients. Precipitates chelate metal ions, vitamins, and other media components, effectively reducing their bioavailability and altering intended media composition.
๐Ÿ’ก 4 ยท โœ“ 4
moderate
Photooxidative Cell Death from Fluorescent Light Exposure
Cells cultured near fluorescent lights or windows show increased death rates, membrane blebbing, and oxidative stress markers. HEPES-buffered media particularly affected.
๐Ÿ’ก 4 ยท โœ“ 5
moderate
Contact Inhibition and Nutrient Depletion Cell Death
Cultures exceeding 80-100% confluence show central necrosis, detachment of cell sheets, and debris accumulation. Media becomes acidic (yellow) and depleted.
๐Ÿ’ก 4 ยท โœ“ 5
moderate
Temperature-Induced Protein Precipitation in Culture Media
Turbidity or visible precipitates appear in cell culture media after temperature changes. High-molecular weight plasma proteins fall out of solution, creating visible cloudiness that may interfere with microscopy and imaging assays.
๐Ÿ’ก 4 ยท โœ“ 4
moderate
Replicative Senescence After Excessive Passaging
Primary or finite cell lines show progressive slowing of growth, enlarged flattened morphology, failure to reach confluence, and increased spontaneous death after many passages.
๐Ÿ’ก 4 ยท โœ“ 5
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