Staining for Transcription Factors (e-Bioscience)
eBioscience Cytofix/Fixation/Permeabilization Concentrate (cat. 00-5123): 30 ml. Store at 4°C. eBioscience Fixation/Permeabilization Diluent (cat. 00-5223): 100 ml. Store at 4°C. Dilute 1 part Concentrate with 3 parts Diluent to make the Fix/perm buffer. eBioscience Permeabilization/Wash Buffer (10X) (cat. 00-8333): 100 ml. Store at 4°C. Dilute to 1X with deionized/distilled water and store at 4°C.
The Foxp3 Staining Buffer (00-5523-00) has been formulated and optimized for staining against Foxp3 and also other transcription factors such as Nanog, Tbet, Gata-3, Ror gamma as well as cytokines.
Do surface staining normally. Use (when possible) 1 – 5×106 cells/wells.
Prepare the Fix/perm buffer by diluting 1 part Concentrate with 3 parts Diluent. Add Fix/perm buffer in 200ul for 30 min on ice.
Wash 1x in FACS buffer.
Prepare the Permeabilization/Wash Buffer by diluting to 1X with deionized/distilled water. Wash twice in 200ul Perm/Wash Buffer.
Incubate 30min in 50ul FC block at 1/100 with 5% normal mouse serum (NMS) in Perm/Wash Buffer.
Incubate with anti-transcription factors Ab for 30min on ice diluted in Perm/Wash Buffer. To do so, without washing add 50ul Perm/Wash Buffer containing either of the following antibody at the given concentration.
+ Foxp3 Pacific Blue 1/25
+ Helios Pacific Blue 1/10 (weak)
+ Helios PE 1/20
+ GATA-3 PE 1/25
+ GATA-3 Alexa Fluor 600 1/15
Wash 2x in 200ul Perm/Wash Buffer.
Wash 1x in FACS buffer. Staining is ready and you can go to the flow cytometer.
Staining for Transcription Factors (e-Bioscience) —> PDF