We recommend users to visit 'Current Protocols in Cytometry' to find detailed protocols for most applications. The facility has a license to access these protocols and can share with facility users.

Cell Cycle

Below is a commonly used protocol for cell cycle analysis using Propidium Iodide.  Please contact us with questions as these are  basic procedures which can be modified to fit specific applications.

Cell Cycle Protocol

Open access paper on analysis of cell cycle by flow cytometry: Adv Exp Med Biol 2010. Important considerations when staining for DNA in cells.

Fixation - all human and non-human primate materials must be fixed or run on a dedicated BSL-2 instrument (EHS Schedule F required).

There are a number of fixation protocols, depending on cell types. We recommend using 1% Formalin (final concentration), EM Grade Formalin from Ted Pella (#18505, which comes in 10x10 mL ampules of 16% Formalin). Cells should be resuspended in PBS prior to fixation to prevent fixing cells to each other. Gently swirl or vortex cell suspension while adding and equal volume of 2% Formalin in a drop wise fashion. Purchasing formalin in solution reduces lab worker exposure to fumes that occur when preparing paraformaldehyde.You can dilute the 16% Fomalin in PBS and keep it in the refrigerator in a capped tube for several weeks without losing activity.

For an explanation of the differences between Paraformaldehyde, Formalin and Formaldehyde, refer to this Technical Note from Pelco.

Titering Antibodies

The Flow Cytometry Core staff highly recommends that you titer your antibodies prior to use in important experiments. This will both save you money and improve the quality of your data. You can find detailed instructions on how to titer your antibodies in this Current Protocols in Cytometry 54:6.29.1 - 6.29-9 (2010) Article by Rudd Hulspas:

Titration of Fluorochrome-Conjugated Antibodies for Labeling Cell Surface Markers on Live Cells.

DNAse to prevent cell clumping

We recommend having 25-50 ug/mL of DNAase (or 1-5 mM) in the presence of 1 mM Magnesium (e.g. not good to combine with EDTA).  A full protocol can be found on the UCSD website: http://cancer.ucsd.edu/research-training/shared-resources/flow-cytometry/protocols/Pages/dnase-i.aspx

Quality Control

March, 2011 - we are encouraging all analyzer users to take control of their quality control! We recommend each lab have available some Spherotech 8-peak Rainbow Calibration beads, Cat # RCP-30-5A. You can see the recommended set up in their pdf here: Rainbow Calibration Particles.

LSR II users should be running CS&T beads from BD before each experiment.

Tubes for CyAn samples less the .5 mL: Biorad  # 223-9391 or Fisher # 02-681-376