Freshly isolated human TFH and non-TFH cells and
mouse naïve and follicular T (IL-21+ or IL-21-)
cells were stimulated with 10 μM forskolin (Sigma) for 24h in RPMI 1640
medium supplemented with 3% BSA, 50 μg/ml D-glucose (Sigma), 2 mM
L-glutamine, 100 U penicillin-streptomycin, 0.1 mM non-essential amino acids and
100 mM Hepes (Gibco, Thermo Fisher Scientific). Cells were then collected,
prefixed with 50 mM cacodylate and 1% sodium metabisulfite (MBS, Sigma) and
fixed with 5% glutaraldehyde (GA) in 0.1 M cacodylate and 1% MBS for 15 min at
RT. After washing with 1% MBS in 50 mM Tris (Tris–MBS) twice, the cells
were incubated with or without rabbit anti-dopamine pAb (Millipore) in
Tris–MBS containing 0.05% Triton-X for 1h at RT, followed by Alexa Fluor
488-conjugated anti-rabbit IgG (Invitrogen) for 30 min at RT. After washing with
Tris–MBS, samples were mounted on cover glasses and visualized with a
Zeiss Axio Observer microscope and then were analyzed with a LSR Fortessa
(BD).
mouse naïve and follicular T (IL-21+ or IL-21-)
cells were stimulated with 10 μM forskolin (Sigma) for 24h in RPMI 1640
medium supplemented with 3% BSA, 50 μg/ml D-glucose (Sigma), 2 mM
L-glutamine, 100 U penicillin-streptomycin, 0.1 mM non-essential amino acids and
100 mM Hepes (Gibco, Thermo Fisher Scientific). Cells were then collected,
prefixed with 50 mM cacodylate and 1% sodium metabisulfite (MBS, Sigma) and
fixed with 5% glutaraldehyde (GA) in 0.1 M cacodylate and 1% MBS for 15 min at
RT. After washing with 1% MBS in 50 mM Tris (Tris–MBS) twice, the cells
were incubated with or without rabbit anti-dopamine pAb (Millipore) in
Tris–MBS containing 0.05% Triton-X for 1h at RT, followed by Alexa Fluor
488-conjugated anti-rabbit IgG (Invitrogen) for 30 min at RT. After washing with
Tris–MBS, samples were mounted on cover glasses and visualized with a
Zeiss Axio Observer microscope and then were analyzed with a LSR Fortessa
(BD).