The DNA binding ability of NF-кB was quantified by EMSA using NF-кB EMSA kit (Viagene, China) according to the manufacturer's instruction: briefly, the TNC samples were diluted using PBS into 5 μg/μL and subjected to the reaction solution before addition of biotin-labelled NF-кB probe.
Serum BLyS levels were determined by ELISA (R&D Quantikine KIT) in all 73 patients while sTACI (R&D Quantikine, DuoSet) in 60 of them, according to the manufacturer's instruction; briefly (1) for BLys determination, the specified amount of patients' or HI serum was added to a 96-cell microplate already coated with a capture antibody.
At the beginning of the procedure, the participants were given written instructions briefly presenting the context of the study and detailing the task to be performed.
At the beginning of the experiment, the participants were given written instructions briefly presenting the context of the study and detailing the task to be performed.
The manufacturer's instructions briefly describe which type of sample can be analyzed, warning users of the possibility of false results.
In the field, tests were carried out according to the manufacturer's instructions; briefly, 10 μl of serum was added to a test cassette, followed by 2-3 drofs of buffer (provided in the test pack).
Gene expression quantitation was performed according to the manufacturer's instructions; briefly a total of 20 μL of PCR mix containing TaqMan Universal PCR master mix with AmpliTaq Gold DNA Polymerase, TaqMan Gene Expression Assay mix, and 4 ng of cDNA was used.
The reaction was performed according to the manufacturer's instructions, briefly, 30 μl of RNA was mixed with 3 μl of Buffer A, 0.5 μl RNAsin® Ribonuclease inhibitor (20 40 U/μl, Promega) and 1 μl Terminator (1 U/μl).
Etest was performed according to the manufacturer's instructions Briefly, each 150-mm petri plate containing 60 ml of medium was inoculated by streaking the swab over the entire surface of the medium.
For the TUNEL reaction, the in situ Cell Death Detection Kit (Cat. No 1684809, Roche Diagnostics, Mannheim, Germany) was used according to the manufacturer's instructions: Briefly, deparaffinized sections were pretreated with proteinase K in 10 mM Tris/HCl and washed in phosphate buffered saline (PBS).
Cells were transfected using Lipofectamine 2000 in accordance with the manufacturer's instructions: briefly, subconfluent cells were transferred into Optimem-1 for transfection and incubated with a mixture containing 0.7 μg of DNA and 4 μl of Lipofectamine 2000 for 5 h.
