Perm wash 1

Single-cell suspensions were transferred into a 96-well V-bottom plate and washed once with 100 μl FACS buffer (0.5% BSA) in PBS). After spinning, supernatants were removed and cell pellets were resuspended in 50 μl staining solution containing fluorescence-conjugated primary Ab diluted in FACS buffer and incubated for 20 min at RT. Cells were then washed twice and either acquired immediately in a FACS Calibur flow cytometer (BD Biosciences) or BD LSR Fortessa X20 (BD Biosciences). Alternatively, cells were fixed with a prewarmed fixation solution (BD Cytofix, BD Biosciences) for 10 min at 37 °C. Cells were then washed twice in 150 μl permeabilization buffer (BD Perm/Wash I, BD Biosciences), resuspended in 150 μl permeabilization buffer, and incubated at 4 °C for 30 min. Primary Abs, diluted in permeabilization buffer, were added to the cells for 1 h, followed by three washes in permeabilization buffer and the addition of the corresponding secondary Abs (in permeabilization buffer). After three washes, cells were analyzed in a FACS Calibur flow cytometer or BD LSR Fortessa X20. Acquired data were analyzed by FlowJo (FlowJo Software part of BD). Counts, percentages, or median intensity fluorescence values were extracted from FlowJo as excel files.

0.5 Mio cells were harvested and washed twice with 1ml FACS buffer (PBS + 2% FCS). Cells were fixed with 150μl pre-warmed fixation solution (BD Cytofix®, BD Biosciences) for 10 min at 37°C. For staining of intracellular antigens, fixed samples were washed twice in 150μl permeabilization buffer (BD Perm/Wash I, BD Biosciences), re-suspended in 150μl permeabilization buffer, and incubated at RT for 30 min. Permeabilized cells were stained in 50μl permeabilization buffer containing the respective antibody dilution. For fluorescent-conjugated primary antibody staining, samples were incubated for 2h at 4°C. When fluorescent-conjugated secondary antibodies were used, they were diluted in 50μl permeabilization buffer and added to cells for 30 min at RT in the dark. Cells were washed 3 times with 1ml permeabilization buffer after each staining and twice with 1ml FACS buffer before surface staining. For staining of surface-expressed LNGFR, mouse anti-LNGFR-PE was added in 50μl of FACS buffer and stained for 30 min at RT in the dark. Cells were washed 3 times in FACS buffer, and samples were acquired at a LSRFortessa flow cytometer (BD Biosciences). The following antibodies were used:

Antigen	Species	Fluorochrome	Clone	Supplier
HA	Rabbit	AF488	C29F4	CST
LCK	Mouse	n.a.	3A5	Santa Cruz Biotechnology
Mouse IgG (H+L)	Goat F(ab’)2	AF647		CST
LNGFR (CD271)	Mouse	PE	ME20.4-1.H4	Milteny Biotec