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

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