KOSEN 한인과학기술자네트워크

적당한 buffer(제 경우는 PBS w/ 2% FBS)에 PI를 넣어서 준비해 두신 다음 (저희는 ml 당 8 ul를 사용합니다), 마지막 wash 가 끝난 후 세포를 resuspend 할 때 이 버퍼를 사용하시면 됩니다. 간혹 PE를 쓰시는 경우에는 PI가 겹치기 때문에 잘 되지 않으므로, (due to the extensive overlap of the emission spectra) 7-amino-actinomycin D를 대신 사용하시는 게 좋습니다. >PI single staining으로 cell death를 FACS로 살펴보고 싶어서요~~ >근데 프로토콜을 몰라서 프로토콜 부탁드립니다~~ >제 생각에는 그냥 cell collect 후에 washing하고 >바로 PI solution 넣어서 FACS 찍으면 되지 않을까 싶은데.... >그렇게 하면 되나요??

PROPIDIUM IODIDE STAINING OF DEAD CELLS FOR FLOW CYTOMETRY Propidium iodide (PI) intercalates into double-stranded nucleic acids. It is excluded by viable cells but can penetrate cell membranes of dying or dead cells. I. MATERIALS: 1. Propidium iodide (e.g., Cat #537059, Calbiochem, San Diego, CA) 2. Buffer: 1 X PBS (Ca2+ and Mg2+ free, e.g., Cat #9240, Irvine Scientific, Santa Ana, CA) +2% newborn calf serum (or 0.2% BSA) +0.1% sodium azide A) PI buffer: Dissolve PI in buffer at a concentration of 1 microgram/ml. Keep the solution tightly closed at 4°C protected from light. Discard after 1 month. B) PI stock buffer: Dissolve PI in buffer at a concentration of 500 micrograms/ml. Keep the solution tightly closed at 4°C protected from light. We have kept this solution for several months and did not observe loss in staining activity. II. METHOD: Stain your cells as outlined in the protocol for single-color staining with FITC-labeled monoclonal antibodies. A) After the last washing step resuspend your cell pellet in the PI buffer and keep your samples in that solution at 4°C protected from light until analysis on the flow cytometer. B) After the last washing step resuspend your cells as usual for analysis. If you want to assess viability of your samples add 2 microliters of the PI stock solution to each tube and mix well. Keep the samples in this solution at 4°C protected from light until analysis on the flow cytometer. NOTE: This method cannot be used on formaldehyde-fixed samples. It is possible to use it on samples that are stained with PE (phycoerythrin)-conjugated antibodies according to a method by Sasaki et al. (Cytometry 8:413, 1987). However, because of the extensive overlap of the emission spectra of PI and PE it is preferable to use dead cell discrimination with 7-amino-actinomycin D (see the appropriate protocol).

1. Wash cells with PBS 2.Treat 200 ul of Trypsin-EDTA 3.Harvest all of cells and add 2ml media 4.Centrifuge 1200 rpm, 2 min (Depend on cells, it would be chagne the rpm). 5.Remove the medium and wash the cell pellet with PBS. 6.Centrifuge 1200 rpm, 2 min 7.Add 750 ul of PBS and keep the tubes on the ice. 8.Before measuring FACS, add 250 ul of PI (200 ul/ml).