LIVE/DEAD™ Fixable Blue Dead Cell Stain Kit, for UV excitation

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Add 1 μL of the reconstituted fluorescent reactive dye and incubate at room temperature or on ice for 30 minutes, protected from light Add 100 μL of 37% formaldehyde and incubate at room temperature for 15 minutes

Protocol tips
Add 1 μL of the reconstituted fluorescent reactive dye and incubate at room temperature or on ice for 30 minutes, protected from light Add 100 μL of 37% formaldehyde and incubate at room temperature for 15 minutes

Publication protocol

Surface marker expression was analyzed using the following antibodies: CD3 (clone 17A2) and B220 (clone RA3-6B2) (all from eBiocience according to data sheet). Staining was performed in the presence of Fc-receptor blocking antibody (clone 2.4G8, a kind gift of E. Kremmer, Helmholtz Zentrum München). Intracellular cytokine staining for IFN-ɣ (eBioscience Clone XMG1.2) was performed as follows: 1x106 cells/ well were put in 200μL of stimulation media (RPMI 1640 + 10% FCS supplemented with PMA (20ng/mL), Ionomycin (1μg/mL) and Brefeldin A (10μg/mL)) in a 96 well flat bottom plate and incubated for 4h at 37°C. Unstimulated cells were handled in parallel, without PMA/Iono stimulation but with Brefeldin A. After incubation, cells were washed twice with PBS and used for surface marker staining (B220) followed by intracellular CD3 and IFN-ɣ staining with Fix/Perm-Kit (eBioscience) as described by the manufacturer.

All cells were processed on a LSRII Fortessa Flow Cytometer (BD) and analyzed with FlowJo9.6.2 software. Dead cells were excluded using Live/Dead Fixable Blue Dead Cell Stain Kit (Invitrogen) and doublets by gating on single cells.

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