Gear|Products|Items|Tools|Supplies|Devices|Machines|Accessories|Stuff|Goods|Gadgets|Outfits|Things|Materials|Objects|Appliances|Resources|Solutions|In

inactivation of
?uorophores to progressively build a multichannel picture. Because Gear|Products|Items|Tools|Supplies|Devices|Machines|Accessories|Stuff|Goods|Gadgets|Outfits|Things|Materials|Objects|Appliances|Resources|Solutions|Instruments|Components|Focus|Units} And Construction Throughout New York --Ruxolitinib Leaves Without Any Regards CycIF utilizes conventional
reagentsandinstrumentationandisnomoreexpensivethanconventionalIF,itissuitablefor
high-throughputassaysandscreeningapplications.

1HMSLINCSCenter&LaboratoryofSystemsPharmacology,HarvardMedicalSchool,Boston,Massachusetts02115USA.2DepartmentofSystemsBiology
HarvardMedicalSchool200LongwoodAvenue,Boston,Massachusetts02115USA.Correspondenceandrequestsformaterialsshouldbeaddressedto
P.K.S.(email:peter_sorger@hms.harvard.edu).
NATURE COMMUNICATIONS |6:8390|DOI: 10.1038/ncomms9390 |www.nature.com/naturecommunications
1
&2015MacmillanPublishersLimited.Allrightsreserved.



ARTICLE
NATURECOMMUNICATIONS|DOI:10.1038/ncomms9390
I
ncreasing the multiplicity of single-cell measurement (the multichannel image.

We also describe several routines for
number of independent measurements performed on each processing multichannel images, which is a ?eld unto itself,
cell) has the potential to reveal interdependencies among Gear|Products|Items|Tools|Supplies|Devices|Machines|Accessories|Stuff|Goods|Gadgets|Outfits|Things|Materials|Objects|Appliances|Resources|Solutions|Instruments|Components|Focus|Units} And Show In Latin America - -Minoxidil Actually Leaves Without Any Good-Bye but a wide variety of software methods should be equally good;
differentiationstatus,signal-transductionstate,localenvironment http://loci.wisc.edu/software/homeprovidesagoodintroduction.
andphenotypethatarenotevidentwhenthesamemeasurements
1�C4
aremadeindependentlyorperformedatthepopulationlevel
.
Results
Multiplex data on cell-to-cell ?uctuations can also be used to
characterize signalling pathways in new ways, as illustrated by
Conceptual overview of cyclic immuno?uorescence.

A typical
5-cycle CycIF procedure yields 16-channel images of slides or
platesatacostofB$2/sample(ina384-wellplate)andcanbe
performedattheendofFP-basedlive-cellimaging.Thereisno
theoretical Gear|Products|Items|Tools|Supplies|Devices|Machines|Accessories|Stuff|Goods|Gadgets|Outfits|Things|Materials|Objects|Appliances|Resources|Solutions|Instruments|Components|Focus|Units} And Show In Las Vegas --Regorafenib Has Left With No Hasta La Vista limit to the number of CycIF cycles and channels.
However, 8�C16 channel images already present an analytical
challenge and most of our work is therefore concentrated on
validating different panels of antibodies for 4�C5 cycle CycIF. In
this paper we demonstrate the application of CycIF to drug
responseatasingle-celllevelandshowthatmultiplexsingle-cell
measurement reveals new aspects of drug sensitivity and resis-
5,6
methodssuchasWanderlustandviSNE .Relativeto?ow-based
3
methods, in which 430 channels can be recorded per cell ,
immuno?uorescence(IF)istypicallylimitedto4�C6channels.

A
compensatory advantage of imaging is that it reports on spatial
features such as cell shape and protein localization and can be
applied to living cells using dyes and genetically encoded
reporters. Many technologies have been developed to increase
the multiplicity of IF microscopy, including infrared-shifted
7�C10
?uorophores, quantum dots and bar coding
, but these
techniques usually require special reagents or instrumentation,
suchashyperspectralmicroscopes,whicharenotwidelyavailable
andhavetheirownlimitations.Withonechannelusedforimage
segmentationandregistration,we?ndthat3�C4datachannelsisa
typical limit for robust, high-throughput IF imaging in 96- and
384-well plates, particularly when signals are relatively weak or
?uorescentproteins(FPs)arebeingimaged(mos