The disappearance of NC9-containing patches with permeabilization using a detergent indicates that they are very sensitive to any changes in plasma membrane integrity and that FXIIIA could be somewhat weakly associated with the plasma membrane.
Following washes with PBS, permeabilization using 6 N HCl for 7 min was done, which were then neutralised with 0.1 M Borate buffer (pH 8.5) [31].
Following surface staining, cells were incubated with PE-conjugated anti-IL-17A (BD Pharmingen™, USA) after fixation and permeabilization using the IntraPrep Permeabilization Reagent (Beckman Coulter Inc., France).
The membrane permeabilization studies using PI were done on gram positive, B. subtilis.
Cells were washed with FACS buffer before fixation and permeabilization using the BD Cytofix/Cytoperm™ (BD, San Diego, CA, USA) according to the manufacturer's protocol followed by intracellular staining.
For the staining of Chop and T-bet, T cells were fixed with the Transcription Factor Fixation and Permeabilization Kit (eBioscience), using the recommended protocol from the vendor.
Intracellular staining with anti-IL-10 and anti-Foxp3 antibodies was performed after fixation and permeabilization, using the cytofix/cytoperm (BD Bioscience, Le Pont de Claix, France) and Foxp3 Fixation/Permeabilization kits (eBioscience), respectively.
Permeabilization was performed using a FOXP3 fixation/permeabilization kit, also from eBioscience.
Permeabilization was achieved using 0.5% Triton X-100/PBS for 20 min and cells were washed twice with 3% BSA/PBS.
Permeabilization was done using 0.1% triton-X-100 at RT for 5 min, followed by washing again with PBS.
Permeabilization was performed using methanol 50% in PBS.
