Cell Culture Protocols

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Cell Culture Protocols

• The Dos and Donts of cell culture
• The Dos
• Use personal protective equipment
• Wear dedicated PPE for tissue culture facility
  and keep separate from PPE in the general lab
  environment.
• Keep all work surfaces free of clutter.
• Correctly label reagents including flasks,
  medium.
• Only handle one cell line at a time.
• Clean the work surfaces with a suitable
  disinfectant (e.g. 70 ethanol).
• Wherever possible maintain separate bottles of
  media for each cell line.
• Examine cultures and media daily for evidence of
  gross bacterial or fungal contamination.
• Quality control all media and reagents prior to
  use.
• Ensure that incubators, cabinet, centrifugess and
  microsocpes are cleaned.
• Test cells for mycoplasma on a regular basis.

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• The Donts
• Do not continuously use antibiotics in culture
  medium.
• Dont allow waste to accumulate particularly
  within the incubators and culture area.
• Dont have too many ppl in the lab at any one
  time.
• Dont handle cells from unauthenticated sources
  in the main cell culture suite.
• Avoid keeping cell line continually in culture
  without returning to frozen stock.
• Avoid cell culture becoming fully confluent.
  Always sub-culture 70-80 confluency or as
  advised on ECACCs cell culture data sheet.
• Dont allow media to go out of date.
• Avoid water baths dirty.
• Dont allow essential equipment to become out of
  balibration.

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Subculture of Suspension Cell Lines

• Materials
• Media pre-warmed to 37ºC (refer to the ECACC
  Cell Line Data Sheet for the correct medium)
• 70 Ethanol in water
• PBS
• Procedure
• View cultures using a inverted phase contrast
  microsocpe.
• Subculture while the culture media turns to
  yellow.
• Take a small sample of the cells from the cell
  suspension. Calculate cells/ml, then re-seed the
  desired number of cells into freshly prepared
  flasks. The data sheet will give the recommended
  seeding densities.
• Repeat this every 2-3 days

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Subculture of Adherent Cell Lines

• Materials
• Media pre-warmed to 37ºC (refer to the ECACC
  Cell Line Data Sheet for the correct medium)
• 70 ethanol in water
• PBS without Ca2/Mg2
• 0.25 trypsin/EDTA in HBSS, without Ca2/Mg2
• Procedures
• View cultures using an inverted microscope.
• Remove spent medium.
• Wash the cell monolayer with PBS.
• Pipette trypsin/EDTA onto the washed cell
  monolayer.
• Return flask to the incubator and leave it for
  2-10 min.
• Examine the cells to ensure that all the cells
  are detached.
• Resuspend the cells in a small volume of fresh
  serum-containing medium to inactive trypsin.
• Transfer the required number of cells to a new
  labeled flask containing medium.
• Repeat this for every 2-3 days.

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Total RNA extraction

• Protocol
• Centrifuge cells at 1,000 rpm for 5 mins to get
  the cell pellets. Remove medium and wash cell
  pellets with ice-cold PBS for 2 times.
• For less than 1x107 cells, add 1 ml Trizol and
  mix well.
• Add 0.2 ml chloroform and mix well. Sit on ice
  for 5-10 mins.
• Centrifuge at max. speed for 10 mins at 4?.
• Transfer the colorless upper aqueous phase to a
  new tube. The volume of the aqueous phase is
  about 60 of the volume of Trizol.
• Use 0.5 ml of isopropyl alcohol per 1 ml of
  Trizol for RNA precipitation. Incubate samples at
  -80 ? for further purification.
• Note
• Use personal protective equipment (e.g. gloves,
  lab-coat, mask)
• Keep RNA samples at 4 ?.
• Handle RNA samples with RNase free tubes and
  tips.
• Use DEPC H2O for RNA samples.

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RNA purification

• Centrifuge at Max speed for 30 mins at 4C
• Wash pellet in 70 EtOH (in DEPC H2O) for 2 times
• Re-suspend pellet in 100 ul DEPC H2O
• Run Qiagen RNeasy Mini kit
• Add 350 ul RLT into 100 ul sample mix well
• Add 250 ul EtOH (95-100) mix well
• Apply sample (about 700ul) to column, spin at max
  speed for 15 second
• Add 350 ul RW1 into column, spin at max speed for
  15 second
• Add 10 ul DNase I (RNase free DNase) into 70 ul
  RDD
• Pipette 80 ul DNase I solution directly on the
  membrane, place at RT for 15-20 min
• Pipette 350 RW1 into column, spin at max speed
  for 15 second
• Add 500 ul RPE into column, spin at max speed for
  15 second
• Add another 500 ul RPE, spin at max speed for 2
  min
• Transfer column to a new collection tube, add
  30-50 ul DEPC H2O on the membrane, at RT for 5-10
  min, spin at max speed for 1 min.
• Check OD and run gel for the RNA quality.

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