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发表于 2011-12-28 15:45:35
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chujindong 发表于 2011-12-28 11:56
我看几年前国内有人发表中文文章，用的试剂还不是进口的，都是自己配得。 ...
给你一个flow cytometry protocols一书第73页的protocol：
2.3. Surface + Intracellular Staining (Saponin Protocol)
1. Reagents:- 1X PBS (phosphate-buffered saline): Dissolve 1.44 g of Na2HPO4, 0.24 g of KH2PO4, 8 g of NaCl, and 0.2 g of KCl in 850 mL of distilled water. Adjust the pH to 7.4 with HCl and volume to 1 L. Store at room temperature.
- Fetal calf serum (standard cell culture FCS). Store at 4°C.
- Formaldehyde: Paraformaldehyde stocks of 4–36% can be made or bought (we use 16% paraformaldehyde stocks, cat. no. 15710 from Electron Microscopy Sciences, Washington, PA). See Note 2 for preparation. Store at room temperature.
- Staining media: 1X PBS, 4% FCS, and 1 mM sodium azide. Store at 4°C. (See Note 3).
- EDTA: Make a 5 M stock solution and use in preparation of the PBS–EDTA buffer. EDTA is used to avoid cell clumps during flow cytometer acquisition. Store at room temperature.
- PBS–EDTA: PBS and 1 mM EDTA. Store at room temperature.
2. Saponin: Make a 10% saponin stock solution by mixing 10 g of saponin(containing ≥25% saponingen content, from Sigma, St. Louis, MO) with 100 mL of PBS. Place at 37°C until saponin has dissolved with mild stirring. Sterile filter (0.22 μL) and store at 4°C (see Note 4).
3. Phospho wash buffer: PBS, 1 mM β-glycerol phosphate, 1 mM sodium orthovanadate, 1 μg/mL of microcystin (500-μg vials can be purchased through Calbiochem, now EMD Biosciences, Inc., San Diego, CA), and 1 mM azide. This is the base buffer for all subsequent buffer formulations. Store at 4°C.
4. Saponin permeabilization buffer: Phospho wash buffer, 0.2% saponin, 4% FCS, and 1 mM azide. Store at 4°C. Final saponin concentration for permeabilization should be no less than 0.1% per sample. A 0.2% solution is made to account for residual volume in wells left after wash (see Note 5). Store at 4°C.
5. Saponin staining buffer: Same as saponin permeabilization buffer.
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发表于 2011-12-28 11:02:05
