Using Organotypic Tissue Slices as Substrata for the Culture of Dissociated Cells - Methods in Molecular Biology: Extracellular Matrix Protocols - page 239

ICN Biochemicals), 20 n M progesterone, 30 nM sodium selenite, 100 µ M

putrescine, 10 µ g/mL bovine insulin, 1 m M sodium pyruvate, 50 I.U./mL penicil-

lin, 50 µ g/mL streptomycin, and 25 m M HEPES (pH 7.2). Filter sterilize. Store at

4 C. Use preferably within 24 h. Other basal medium and supplements may be

more appropriate for the particular tissue and dissociated cells used.

°

6. Sterile filter paper or gauze. Bleached (white), filter paper from a sealed pack

should be suitable.

2.3. Dissociating Tissue into a Cell Suspension
1. Balanced Salt Solution (BSS): calcium-magnesium-free, Hank's Balanced Salt

Solution. This can be supplemented with 10 m M HEPES, pH 7.2, if good pH
control is needed (BSS will become somewhat basic in room air).

2. Trypsin stock: 5 mg/mL of trypsin from bovine pancreas in BSS. Sterile filter.

Store as aliquots at -20 ° C.

3. DNase stock solution: 1 mg/mL DNase I (grade II, Boehringer Mannheim, Ger-

many) in DMEM or other basal medium. Sterile filter. Store as aliquots at -20 ° C.

4. Trypsin inhibitor: 5 mg/mL trypsin inhibitor (Type I-S) from soybean in DMEM

or other basal medium. Sterile filter. Store as aliquots at -20 ° C.

5. 9" Pasteur pipets with cotton plug. Autoclave to sterilize.

6. 4% BSA: 4% bovine serum albumin (crystalline grade; ICN Biochemicals) in

BSS; bring to pH ≈ 7.3 with 1 M NaOH. Sterile filter. Store as 2.0-2.5 mL aliquots

at -20 ° C.

2.4. Labeling Dissociated Cells
Dye stock solution: Use Cell Tracker™ (Molecular Probes, Eugene, OR)
dyes, such as CMTMR (rhodamine derivative) or CMFDA (fluorescein
derivative). Make 10 m M stock solution in DMSO and store as aliquots at -20

° C.

2.5. Coculture, Fixation, Counterstaining, and Mounting
1. Paraformaldehyde: 4% paraformaldehyde (J. T. Baker, Phillipsburg, NJ) in 0.1 M

sodium phosphate buffer (pH 7.5) or PBS. Paraformaldehyde is toxic, so perform
this procedure in a fume hood. Add paraformaldehyde to water (one half of the
final solution volume), stirring at 65

C. Add 5 M NaOH dropwise until paraform-
aldehyde goes into solution. Bring to the final volume with a 2X stock of buffer
(0.2 M sodium phosphate or 2X PBS). Filter to remove particulate. For paraform-
aldehyde/sucrose, make fix with 0.12 M final sucrose. Add sucrose with the 2X
buffer. Good for 2 wk at 4

°

° C.

2. Bisbenzamide (Hoechst 33258): 1 mg/mL stock solution (100X) in water, then

filter and store at 4 ° C. For use, make fresh by diluting in PBS to 10 µ g/mL.

3. Mounting solution: Saturated sucrose solution containing 0.1% sodium azide

(Fluka AG). Store at room temperature. Other aqueous mounting media may work
as well.

4. Glass microscope slides, 22 × 22 mm coverslips, super glue, and clear nail polish.