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Time

Allow take 3-3.5 hours, add one more hour when seeding on ibidi dishes in the drop

Materials

  • Fibronectin (Sigma; Cat num. F1141-5mg; lot …)
  • Pluronic Acid (Cat bum. 59005, lot P40614-101164)
  • ddH20
  • RPMI (+)
  • KOSR
  • Rock Inhibitor
  • Enzyme T
  • Enzyme T diluent
  • FBS
  • PBS
  • Counting Slide
  • DIshes/slides
    • Ibidi dish with the beads coated surface (dry); 3.14cm2
    • 6well with 2cm stencil; 9.5cm2

Preparation steps

Solutions

Fibronectin

  • Dilute 1mg/mL fibronectin to 50ug/mL in PBS
  • Amount:
    • 0.75mL per dish
    • 2mL per 1w6

Pluroinc Acid

  • Dilute to 1% from 10% stock in ddH20
  • Amount:
    • 3mL per dish
    • 3mL per 1e6 NOTE: filter through 22um filter

Stop solution

  • dilute FBS to 20% in PBS
  • Amount:
    • 2mL per 12well
    • 1mL per 24 well

Replating medium

  • 10% KOSR in RPMI(+), add 1to2000 Rock Inhibitor
  • Amount:
    • 3mL per ibidi dish
    • 3mL for 6well dish
    • 1mL for counting of cells

Enzyme T

  • dissolve 33uL/mL of buffer X
  • Amount:
    • 400uL per 1w12 well
    • 200uL per 1w24 well

Process

Coat and sterilze the plates

  • Ibidi:
    • Add 750uL drop on the ibidi dish, spread to edges using pipette (is very hydrophobic)
  • 1w6:
    • Add 2mL swirl to make sure all surface is coated
  • Incubate 1hr at RT
  • Aspirate FN, Wash with 3mL PBS 3x
  • Add pluronic 3mL (both ibidi or 1w6)
  • Incubate 30min at RT (Ideally start harvesting cells at this point)
  • wash with PBS 3mL 3x

Collect cells

  • wash with PBS
  • add diluted enzyme T (200uL/24 well, 400uL/12 well)
  • incubate 8-10min
  • Add Stop solution (300ul/24well, 600ul/12 well)
  • Collect by pipetting, disrupt to single cells
  • Add (500ul; 1mL) to wash down
  • Spin at 300g for 3-5min
  • Resuspend in 0.5mL replating media (for 24 well, for the rest, 1mL is enough)
  • count

Seed cells

  • create a solution
    • Ibidi: approx 100k cells/mL in replating solution
    • 1w6: approx 30k cells/mL
  • Deposit:
    • Ibidi: as a drop approx 600uL, spread to the edges to fill the whole surface of insert
    • 1w6: 3mL in the dish
  • Add media (Ibidi only):
    • Let the cells to sit down for 1 hour (check after 30 min if its okey), but 1hr is better
    • Fill the rest of the vessel with the replating medium (2mL)

Change media

  • change media day after
  • afterwards cheange media to RPMI (+)
  • change every second day
  • Measure from the day 3 onwards