Protocols

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

 Live-Dead cell discrimination

There are a number of great reagents to gate out your dead cells and improve the quality of your data. Propidium Iodide or 7-AAD are favorites, but can interfere with other channels. An number fixable live-dead dyes are now available 

Life Technologies LIVE/DEAD:   http://www.lifetechnologies.com/us/en/home/references/protocols/cell-and-tissue-analysis/flow-cytometry-protocol/cell-viability/live-dead-fixable-dead-cell-stain-kits.html 

Biolegend Zombie Dyes:   http://www.biolegend.com/live_dead 

 RNA extraction from sorted cells:

 Tighe S, Held MA.  Isolation of total RNA from transgenic mouse melanoma subsets using fluorescence-activated cell sorting.  Methods Mol Biol. 2010;632:27-44. doi: 10.1007/978-1-60761-663-4_2.    

http://www.abrf.org/ResearchGroups/FlowCytometryResearchGroup(FCRG)/Publications/Tigheetal.pdf

 

Great advice from Scott Tighe:  'formaldehyde is the quickest way to degraded RNA. Absolutely the last thing you'd want to expose RNA to. RNA later, RNAZap, RNAaway are also reagents you need to avoid.

'The options are either a direct sort into Trizol or RLT buffer, ethanol, or PBS with an RNase inhibitior, Media with RNase inhibitor.  Trizol LS allows the recovery of all RNA (microRNA, lncRNA, mRNA, rRNA ect). Each as it strengths and weaknesses.There is no "one size fits all"

'Remember when you sort into Trizol LS or RLT use need to extract the same day. do not freeze and extract days later. Not all cells can handle extended exposure to these reagents. Remember the 2'-OH group of RNA is highly reactive to hydrolysis.'

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.

We  encourage 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 below.

 

 

 

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