Hippocampal Slice Culture In hood




Дата канвертавання21.04.2016
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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.

    • 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







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


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