Components of the hybridization buffer and the washing procedure were used according to Szabo et al. (2000).
The beads were washed three times with lysis buffer, and the washed beads were suspended with SDS PAGE sampling buffer.
The nuclei were washed twice with a large volume of lysis buffer, and the washed nuclear pellets were resuspended in freezing buffer (50 mM Tris HCl, pH 8.3, 40% glycerol, 5 mM MgCl2, 0.1 mM EDTA), counted, and stored in 100 μl aliquots containing 5 × 10 nuclei.
The labeled mitochondria were resuspended in fresh respiration buffer and the last wash supernatant was saved.
Wells were washed with 0.5 mL of Dulbecco's phosphate buffered saline and the wash was combined with the cells/media.
Beads were washed with small amount of lysis buffer, and the first two washes were pooled with the eluate.
Detached cells were washed with PBS containing 1% FCS and 1% penicillin/streptomycin (wash buffer), and resuspended in the wash buffer (106 cells/100 μl).
Erythrocytes were lysed with ACK lysis buffer, and the splenocytes were washed 2 times with RPMI supplemented with 10% FCS and seeded at 3×105 per well of a 96 well culture plate.
Thereafter, samples were re-suspended in 50 μl of Beadlyte Cytokine Assay Buffer and the vortex and washing procedures repeated.
The medium was applied to a 1 ml Ni-NTA (Ni2+-nitrilotriacetate) resin (Qiagen) equilibrated in TNC buffer and the resin was washed in the same buffer to remove unbound proteins.
