Reagents

• Magnesium Chloride: Sigma, 100 g, FW 95.21, M8266, $25.15
• Potassium Ferrocyanide: Sigma, 100 g, FW 422.39, P9387, $12.00
• Potassium Ferricyanide: Sigma, 100 g, FW 329.25, P3667, $21.60
• 25% Glutaraldehyde: Sigma, 100 ml, G6257, $20.10
• 5-Bromo-4-Chloro-3-Indolyl α-D-Galactopyranoside (X-Gal) Sigma, 100 mg, FW 408.6, B4252, $59.60
• N,N-Dimethyl Formamide: Sigma, 250 ml, FW 73.10, D4551, $15.35
• 1x PBS: BioWhittaker, 500 ml, 17-515F, $XX

Solutions

• 0.05% glutaraldehyde: add 80 µl 25% glutaraldehyde to 39.92 ml 1x PBS
• 20 mg/ml X-gal: dissolve 100 mg X-gal in 5 ml N,N-DMF
• 500 mM potassium ferrocyanide: dissolve 2.11 g in 10 ml ddH₂0
• 500 mM potassium ferricyanide: dissolve 1.65 g in 10 ml ddH₂0
• 1 M magnesium chloride: dissolve 0.95 g in 10 ml ddH₂0
• X-gal solution: (1 mg/ml X-Gal in 5 mM K₄Fe(CN)₆^.3H₂O/5 mM K₃Fe(CN)₆/1 M MgCl₂/PBS): add 1 ml 20 mg/ml X-gal, 200 µl 500mM potassium ferrocyanide, 200 µl 500mM potassium ferricyanide, 20 µl 1 M MgCl₂ to 18.58 ml 1x PBS

Protocol

1. Aspirate media.
2. Add 2 ml 0.05% glutaraldehyde.
3. Incubate 5 minutes at RT.
4. Discard and put in proper waste (DO NOT ASPIRATE INTO BLEACH).
5. Add 2 ml PBS. Aspirate.
6. Add 2 ml PBS.
7. Incubate 10 minutes at RT. Aspirate.
8. Add 2 ml PBS. Aspirate.
9. Cover cells with X-gal solution.
10. Incubate at 37°C/5% CO₂ for 2-24 hours. Positive cells should turn blue.

Note: Can be kept at 4°C wrapped in Saran Wrap.

For specifics on appropriate handling and waste procedures please see the online chemical SOPs or our waste and spill notebook located in room 352.

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