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
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