Using Organotypic Tissue Slices as Substrata for the Culture of Dissociated Cells - Methods in Molecular Biology: Extracellular Matrix Protocols - page 241

1.5 h) in medium on
ice until all slices collected. Culture in a humidified atmosphere at 37

disks on parafilm). Slices can be stored for short periods ( ≈

° C ( see

Note 15 ).

3.3. Dissociating Tissue into a Cell Suspension
1. Dissect tissue for dissociation and place in 1 mL BSS in a 15-mL polypropylene,
conical tube ( see Note 16).
2. Add 45 L DNase (final concentrations of

0.2 mg/mL and 0.09 mg/mL, respectively). Incubate for

µ L of the trypsin stock and 100 µ

≈ 10 min at 37 ° C with

occasional, gentle mixing ( see Note 17 ).

3. Add another 250 L of the

trypsin inhibitor. These additions raise the final DNase concentration to 0.2 mg/mL
and the inhibitor to 0.3 mg/mL. Incubate for another 2-5 min at 37

µ L DNase, 300 µ L of complete medium, and 100 µ

° C.

4. Make a trituration pipet by flame-polishing the tip of a Pasteur pipet so that it

tapers to a hole 0.2-0.7 mm in diameter ( see Note 18 ). To prevent loss of cells

on the glass, coat the inside of the pipet with 4% BSA or serum just before use.
5. Gently triturate the tissue by drawing and ejecting the entire tissue suspension in
and out of the pipet ( see Note 19 ). To avoid introducing bubbles into the suspen-
sion, eject it from the pipet along the wall of the tube at a height above the final
liquid level. Repeat until the most of the tissue has dissociated (about 5X, see
Note 20 ).
6. Add ice cold BSS to bring the final volume to 10-12 mL, mix, and allow
the undissociated tissue to settle by gravity, while keeping the tube on ice
(5-10 min). Transfer the cell suspension to a new tube and discard the settled chunks.

≈

7. If cells are to be labeled, go to Subheading 3.4.1. If cells are to be plated directly,

go to Subheading 3.4.4.

3.4. Labeling Dissociated Cells
1. Make fresh labeling medium by diluting the dye stock in warm complete medium

to a final concentration of 1-25 M ( see Note 21 ). Centrifuge the labeling

medium to remove undissolved dye and filter sterilize before use.

µ

2. Centrifuge the cell suspension (100-300 g , 10 min, 4 ° C) and gently resuspend in

0.5-2 mL labeling medium. Incubate at 37 C for 30-50 min with occasional mix-

ing to resuspend settled cells ( see Note 22 ).

°

3. Add cold BSS to bring final volume to 10-12 mL and mix.

4. Below the cell suspension, underlay a 2-2.5 mL cushion of cold, 4% BSA. Place

the tip of a Pasteur pipet, filled with BSA solution, at the bottom of the tube and
slowly eject the solution so as not to disturb the cushion as it forms ( see Note 23 ).
Centrifuge (150-300 g , 10 min, 4

° C).

5. Resuspend the pellet in complete medium and dilute the cell suspension to the

appropriate density for plating ( see Note 24 ).

3.5. Coculture, Fixation, Counterstaining, and Mounting
1.

Carefully aspirate or pipet the medium off the slices and add the cell suspension.
Alternatively, you can add the cells in a small volume directly to the medium