Anti mhc class 1 hla a hla b antibody

Transfected primary keratinocytes (passage 2) were examined for MHC I markers using flow cytometry. Non-transfected keratinocytes served as control. According to standardized protocols, 5000 successfully transfected primary keratinocytes were selected for flow cytometric analysis. For immunolabelling, cells were detached from culture flasks with Dispase II (Provitro, Berlin, Germany) and washed with phosphate-buffered saline (Sigma Aldrich, Darmstadt, Germany) containing 10 % FBS. Then, cells were blocked with 1% BSA (Sigma Aldrich, Darmstadt, Germany) in PBS for 30 min at 4 °C, centrifuged for 5 min at 300× g, resuspended in PBS, and incubated with the primary antibodies (monoclonal rabbit Anti-MHC class I + HLA-A + HLA-B antibody (abcam, Cambridge, UK), final dilution 1:10, for 50 min at 4 °C. After washing with PBS, cells were incubated with fluorochrome labeled secondary antibodies (Santa Cruz, Heidelberg, Germany), final dilution 1:5, for 45 min at 4 °C, washed and then analyzed. For measurement, a FC500 flow cytometer (Beckman Coulter, Krefeld, Germany) was used.

Total protein extract of primary keratinocytes was made in a radioimmunoprecipitation assay buffer (RIPA) containing 0.3 M NaCl, 1% sodium desoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1% Triton-X-100, 20 mM Tris–HCl (pH 8), and 1 mM ethylene diamine tetraacetic acid (EDTA) supplemented with 1 mM phenylmethyl sulfonyl fluoride (PMSF). A total of 25 µg of each protein sample were separated by electrophoresis on 15% SDS–polyacrylamide gels and then transferred on a polyvinylidene fluoride (PVDF) membrane (Millipore Corporation, Bedford, MA, USA). Immunoblotting was performed with monoclonal rabbit Anti-MHC class I + HLA-A + HLA-B antibody (abcam, Cambridge, UK). Odyssey 680/800 nm secondary conjugates (Li-Cor BioSciences, Lincoln, NE, USA) were used for the quantification of protein expression levels and signals were visualized using the Odyssey Infra-Red Imaging System and software (Li-Cor BioSciences, Lincoln, NE, USA).

To prove successful down-regulation of MHC-I-receptors, indirect immunofluorescence staining was performed on transfected primary keratinocytes after 24 h as well as non-transfected cells as control. Cells were fixed with 4% paraformaldehyde (Sigma Aldrich, Darmstadt, Germany) in PBS (Sigma Aldrich, Darmstadt, Germany), permeated with 0.2% Tritron X-100 (Carl Roth, Karlsruhe, Germany), and blocked with 2% fetal calf serum (Biochrom, Berlin, Germany). The primary antibody used in this study was monoclonal rabbit Anti-MHC class I + HLA-A + HLA-B antibody (abcam, Cambridge, UK). The primary antibody was applied for 1 h, followed by extensive washing with phosphate buffered saline (PBS). Alexa Fluor^® 488 conjugated anti-rabbit antibody (Invitrogen, Carlsbad, CA, USA) was used as secondary antibody. Samples were counterstained with DAPI. Inverse fluorescence microscopy was performed with a Zeiss Axiovert 200 M microscope (Carl Zeiss, Oberkochen, Germany) and associated software.