Ronnett Lab- Amy Kleman

1/17/2005

Hippocampal Slice Culture

-In hood:

  • Add 1mL of culture media to each well of a 6-well plate.
  • Place one 0.4m Millicell insert into each well on top of the culture media.
  • No media should be added directly to the insert – even during feeding
  • Place plate into incubator and allow to equilibrate at 37oC, 95% O2, 5% CO2.
  • Add 3mL of dissection media to as many 10mm Petri dishes as needed
  • 1 lobe/dish
  • Add 10mL of dissection media to one 60mm Petri dish and place directly on ice.

-On benchtop:

  • Install blade of the Vibratome Serious 800 tissue chopper
  • Screw tightly as so blade is always horizontal with the chopping platform
  • Turn thickness dial so each slice is 350m thick
  • Remove head of 8 day postnatal rat pup
  • Remove entire brain from the skull
  • Remove the cerebellum from the brain
  • Split the brain into its right and left hemispheres and place each hemisphere in its own 10mm dish
  • Place one lobe onto ice and prepare the other lobe for dissection
  • Remove lobe from 10mm dish and place on gauze pad to dry any excess media
  • Spray blade of Vibratome with 70% ethanol
  • Place dried lobe unto circular cutting board with the flat side of the love facing you (ie. Olfactory bulb facing the your right)
  • Place cutting board on chopping platform and begin slicing
  • Carefully remove sliced brain from cutting board and place back into its 10mm dish with dissection media
  • Under microscope, use 45o curved forceps to separate the slices about halfway through the lobe.
  • Usually you can see a group of blood vessels in the area where the hippocampus is located – Split the slices here if possible
  • The hippocampus will be cut into about 10 separate slices which are held together via meninges and appear as a “stack of coins”
  • Dissect out as many single slices as you can
  • Usually about 5-6 slices are well enough maintained to plate
  • Use a pipette to gently remove slices from 10mm dish and place into 60mm dish on ice
  • Repeat for as many brains as is necessary for a 6-well plate
  • 1 well = 5 slices
  • About 4 rat pups = 1 full 6-well plate

-In hood:

  • Use pipette to move slices from 60mm dish to 6-well plate inserts
  • After ½ hour, remove any media from the insert that may have been left behind when plating the slices

-Up-keep:

  • Keep slices in incubator (37oC, 95% O2, 5% CO2)
  • Feed slices Mondays, Wednesdays and Fridays by removing all of the culture media in the well via vacuum and adding back 1mL of fresh culture media
  • Slices can be used 14 days after plating when they have flattened to reach a thickness of 100m

-Medias

  • Dissection Media (1L @ pH 7.3)

Ingredient / [Final] / Amount/Volume
Hank’s Balanced Salt Solution / 9.52g
NaHCO3 / 4.2mM / 350mg
HEPES Salt / 10mM / 2.83g
D-(+) Glucose / 33.3mM / 6g
Pen-Strep (100X) / 1X / 10mL
BSA / 0.3% / 300mg
MgSO4-7H2O / 1.44g
  • Check the pH
  • Filter sterilize
  • Culture Media (409mL)
  • Add 12.8g D-(+) glucose to 500mL of Hank’s Balanced Salt Solution
  • Filter sterile

Ingredient / [Final] / Amount/Volume
MEM / 50% / 200mL
Hank’s (as prepared above) / 25% / 100mL
Horse Serum / 25% / 100mL
1M HEPES buffer / 12.5mM / 5mL
Pen-Strep (100X) / 1X / 4mL
  • Check the pH – should be 7.4
  • Filter sterile

-Ordering Information:

Item / Company / Catalog # / Price / Order from…
Hank’s powder / Sigma / H2387 / Core
NaHCO3 / Sigma / S6297 / Core
HEPES / Sigma / H3375 / Core
D-(+) glucose / GIBCO / 15023-021 / Core
BSA / Sigma / A4503 / Core
MgSO4-7H2O / Sigma / M2643 / Core
Horse Serum / Hyclone / SH30074.03HI / Hyclone
MEM / GIBCO / 11575-032 / 15.85 / Core
Hank’s (HBSS) / GIBCO / 24020-117 / 39.80 / Core
1M HEPES buffer / GIBCO / 15630-080 / 39.80 / Core
Pen-Strep (100X) / GIBCO / 15070-063 / 12.85 / Core
Mcllwan Tissue Chopper / Vibratome / Vibratome
6-well inserts / Millipore / PICM03050 / Millipore
6-well plates / Falcon / JHMI
60mm dish / Corning / JHMI
10mm dish / Corning / JHMI
Gauze / Johnson & Johnson
Forceps / FST / 11251-35 / 29.75 / FST