FACS-protocol

Acc.no: 1VBVX85 | Published: 2010-03-21 by jennie

Keywords: FACS, cell analysis, tranfected cells, G1 arrest, cell-cycle arrest, population

Cell cycle distribution and FACS analysis of transfected cells

Required Materials

Cells , to be analysed
Clifton Cyclone vortex
Mini centrifuge
Methanol
Fetal calf serum , FCS
PBS
Trypsin
Nocodazole , 4 mg/ml
Lovastatin , 20 µM
Cell culture media , in accordance with cell type
FACS

Main steps

Seed 250.000 cells in 6 wells and transfect oligoes or plasmids simultaneously.
Wash once in PBS+ 3% FCS (remove by sucktion, notice white pellet, spin…
Seed 250.000 cells in 6 wells and transfect oligoes or plasmids simultaneously.
Change media 6h post transfection and include cell cycle inhibitors (or…
Harvest media and cells by trypsination (into 15 ml tube).
Spin and wash in PBS + 3% FCS
Resuspend i 300 µl PBS + FCS.
Fix by 800 µl -20°C methanol på vortex.
Incubate 30 min on ice or in 4°C ON.
Spin and wash in PBS + FCS (remove by suction, notice white pellet, spin…
(1VBVX4C)
Incubate 30 min at 37°C.

Instructions

FACS analysis.
Wash once in PBS+ 3% FCS (remove by sucktion, notice white pellet, spin 2000 rpm).
Seed 250.000 cells in 6 wells and transfect oligoes or plasmids simultaneously.
Change media 6h post transfection and include cell cycle inhibitors (or at the following day depending on incubation times):
- 20 uM lovastatin (G1 block)
- 4 ug/ml nocodazole (M block) (cells will float)
Nocodazole is a spindle drug that blocks cells in mitosis. It is used to better visualize a possible G1 arrest, since exponential growing cells already have 50-70% of the population in G1
Harvest media and cells by trypsination (into 15 ml tube).
Spin and wash in PBS + 3% FCS
Resuspend i 300 µl PBS + FCS.
Fix by 800 µl -20°C methanol på vortex.
Incubate 30 min on ice or in 4°C ON.
Spin and wash in PBS + FCS (remove by suction, notice white pellet, spin 2000 rpm).
Resuspend in 400 µl PI sol. (50 µg/ml PI, 10 mM Tris pH 7.5, 5 mM MgCl2, 10 µg/ml RNase).
Propidium iodide (PI) is an ethidium bromide analog that emits red fluorescence upon intercalation with double-stranded DNA. Though PI does not permeate viable cell membranes, it passes through disturbed cell membranes and stains the nuclei. PI is often used in combination with a fluorescein compound, such as Calcein-AM or FDA, for simultaneous staining of viable and dead cells. The excitation and emission wavelengths of PI-DNA complex are 535 nm and 615 nm, respectively.
1. For a 10xPI-solution dissolve 0,5 mg/ml PI i 0.038M citrate pH 7.0
  
  Store in a dark place at 4˚C
Incubate 30 min at 37°C.