Regulator of G-protein signalling 2 mRNA is differentially expressed in mammary epithelial subpopulations and over-expressed in the majority of breast cancers

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).

ISSN: 1465-542X