Protocol for intracellular BCL6 staining

Prepare single-cell suspensions of draining and non-dr. LNs in HBSS (or PBS) by standard procedures.

Wash and count cells.

Add 1-2x10⁶ cells in 100µl per well of a 96-well round-bottom plate.

Add 100µl of 1:1000-diluted eFluor780 Fixable Viability Dye (eBioscience) in HBSS (or PBS) (no serum/protein, no azide!). Incubate for 10min on ice.

Spin down and wash plate 1x with 200µl FACS buffer (2% FCS and 0.05% NaN₃ in PBS).

Add 20µl of blocking solution: 1µg/ml anti-CD16/32 (“Fc-block”) and 2% normal mouse/rat serum in FACS buffer. Incubate for 5min on ice.

Add another 20µl of 2x-diluted antibodies including anti-CXCR5-biotin (clone 2G8, BD Biosciences, 1:50 final dilution) on top of the blocking solution (don’t wash in between). Incubate for 45min on ice.

Wash 2x with FACS buffer.

Add 40µl Streptavidin-APC (1:1000 final dilution) per well and incubate for 30min on ice.

Wash 2x with FACS buffer.

Add 100µl Fixation/Permeabilization buffer from the eBioscience FoxP3 staining buffer set per well and incubate for 15min at room temperature.

Add 100µl Permeabilization buffer from the eBioscience FoxP3 staining set per well and spin down.

Wash 1x with 200µl Permeabilization buffer.

Add 20µl of blocking solution: 1µg/ml anti-CD16/32 (“Fc-block”) and 2% normal mouse/rat serum in Permeabilization buffer. Incubate for 5min at room temperature.

Add another 20µl of 2x-diluted anti-BLC6-PE antibodies (clone K112-91, BD Biosciences, 1:50 final dilution) on top of the blocking solution (don’t wash in between). Incubate for 45min at room temperature.

Wash 3x with Permeabilization buffer.

Resuspend cells in FACS buffer.

Reference:

Cutting Edge: Distinct Waves of BCL6 Expression during T Follicular Helper Cell Development.

Baumjohann D, Okada T, Ansel KM.

J Immunol. 2011 Sep 1;187(5):2089-92.