DTT Processing

1. Pick up 4 blood tubes from ACRC processing lab (M1334). Keep tubes at RT. Drop off 1 tube of Buffer 1.
2. Dilute blood 1:1 with room temperature PBS.
3. Carefully layer 2 volumes of diluted blood on top of 1 volume of lymphoprep in 50ml falcon tubes (ex: 25ml blood on top of 12.5ml lymphoprep).
4. Place tubes in aerosol containment buckets, and spin at 800xg (~1900rpm) for 20 minutes at room temp. Turn the brake OFF.

http://www.nature.com/protocolexchange/protocols/2707

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).

________________________________________
From: Allen, Chris
Sent: Wednesday, March 30, 2011 10:47 AM
To: Baumjohann, Dirk
Subject: RE: LSR II

Hi Dirk,