Propidium Iodide for DNA Content<\/p>\n
Note:\u00a0After running samples with Propidium Iodide the cytometer needs thorough cleaning with 10% bleach and distilled water!<\/p>\n
Solutions<\/p>\n
\u00b7\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 70% ethanol<\/p>\n
\u00b7\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 ribonuclease (100 \u00b5g\/ml DNase free, Sigma)<\/p>\n
\u00b7\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 propidium iodide (50 \u00b5g\/ml in PBS)<\/p>\n
Procedure<\/p>\n
1.\u00a0\u00a0\u00a0 Harvest cells. Spin at 1200 rpm for 5 minutes.<\/p>\n
2.\u00a0\u00a0\u00a0 Discard supernatant; add 1 ml ‘ fridge cold’ 70% ethanol to 1×106 cells while vortexing the cell pellet gently.<\/p>\n
3.\u00a0\u00a0\u00a0 Fix for at least 30 min. at 4\u00b0 C.<\/p>\n
4.\u00a0\u00a0\u00a0 Spin at 2000 rpm for 5 min. Once in 70% ethanol, samples may be kept for up to 2 weeks.<\/p>\n
5.\u00a0\u00a0\u00a0 Discard supernatant and resuspend cells in 1 ml PBS. Spin 2000 rpm for 5 min.<\/p>\n
6.\u00a0\u00a0\u00a0 Repeat step 5 twice more.<\/p>\n
7.\u00a0\u00a0\u00a0 Add 100 \u00b5l ribonuclease (100 \u00b5g\/ml, DNase free, Sigma). Incubate at room temperature for 5 min.<\/p>\n
8.\u00a0\u00a0\u00a0 Add 400 \u00b5l propidium iodide (50 \u00b5g\/ml in PBS).\u00a0Note:\u00a0PI is potentially mutagenic so wear gloves.\u00a0Before running the samples stained for DNA content the cytometer’s linearity, resolution and doublet discrimination capability should be verified. DNA QC kit is necessary and should be ordered from Becton Dickinson (Cat. No. 349523). The kit is composed of :<\/p>\n
o\u00a0\u00a0 CEN- chicken erythrocyte nuclei, used in setting cytometer’s photomultiplier tube;<\/p>\n
o\u00a0\u00a0 (PMT) voltages and amplifier gains, and providing information regarding instrument linearity and resolution;<\/p>\n
o\u00a0\u00a0 CTN – calf thymocyte nuclei, a cycling cell component that allows assessment of proper function of doublet discrimination module (DDM) or pulse processing. Please follow the instructions included in the kit for quality control and set-up of the machine.<\/p>\n
9.\u00a0\u00a0\u00a0 Acquire data on a FACScan or FACSCalibur, the cytometer should be triggered on the PI signal. Primary gate is on forward scatter (FSC) against right angle light scatter (SSC).<\/p>\n
10.A secondary gate should be placed around the single cell population on a pulse area versus pulse width dot plot.<\/p>\n
The measurement of cell cycle parameters can be analyzed by modeling programs which fit the best Gaussian distribution curve to each peak, Go-G1, G2-M and then calculate the resulting S-phase. Modfit software from Verity Software House (Verity@vsh.com) or FlowJo can be used.<\/p>\n","protected":false},"excerpt":{"rendered":"
Propidium Iodide for DNA Content Note:\u00a0After running samples with Propidium Iodide the cytometer needs thorough cleaning with 10% bleach and distilled water! Solutions \u00b7\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 70% ethanol \u00b7\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 ribonuclease (100 \u00b5g\/ml DNase free, Sigma) \u00b7\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 propidium iodide (50 \u00b5g\/ml in PBS) Procedure 1.\u00a0\u00a0\u00a0 Harvest cells. Spin at 1200 rpm for 5 minutes. 2.\u00a0\u00a0\u00a0 Discard supernatant; add […]<\/p>\n","protected":false},"author":354,"featured_media":0,"parent":367,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"page-templates\/child-page.php","meta":{"footnotes":""},"class_list":["post-492","page","type-page","status-publish","hentry"],"yoast_head":"\n