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Table 1
Multiple staining protocols for flow cytometric analysis of α-Sca-1/33A10/α-CD45 and α-Sca-1/33A10/α-CD45/α-CD24 stained cells.
Sample	Antibody/antibodies			ToPRO-3 or DAPI

	First incubation	Second incubation	Third incubation

Nonspecific staining control	Rat immunoglobulin	α-rat-FITC	IgG-PE IgG-PE-Cy5 ²IgG-PE-Cy7	No
ToPRO-3 or DAPI control	None	N/A	N/A	Yes
33A10 control	33A10	Anti-rat-FITC	N/A	No
Sca-1 control	α-Sca-1-PE	N/A	N/A	No
CD45 control	^aα-CD45-PE-Cy5 or ^bα-CD45-PE-Cy7	N/A	N/A	No
^bCD24 control	^bα-CD24-PE-Cy5	N/A	N/A	No
Experimental sample	33A10	α-rat-FITC	α-Sca-1-PE ^aα-CD45-PE-Cy5 or ^bα-CD45-PE-Cy7 ^bα-CD24-PE-Cy5	Yes

^aAnti-CD45-PE-Cy5 was used for the four-colour protocol not including CD24. ^bAnti-CD45-PE-Cy7, anti-CD24-PE-Cy5 and the IgG-PE-Cy7 isotype control were only used in the five-colour protocol including CD24 detection. 'Fluorescence minus one (FMO)' controls based on the staining combination used for the experimental sample but in which one antibody was left out and replaced with its isotype control were also used to set sort gates correctly. For simplicity, these have not been shown. Live/Dead cell exclusion used either ToPRO-3 or DAPI (4,6-diamidino-2-phenylindole dihydrochloride).
Smalley et al. Breast Cancer Research 2007 9:R85 doi:10.1186/bcr1834

Table 1