2.6 NF-κB Detection

2.6 NF-κB Detection

2.6.1 Preparation of Single Cell Suspension

Under sterile conditions, take 0.5cm³ of fresh tumor tissue, place it in a petri dish, use phosphate buffered saline (PBS) to remove blood and blood clots from the surface of the tumor tissue, add a small amount of PBS; use ophthalmic scissors to cut the tissue into a homogenous slurry, add 5ml PBS; use a pipette to draw the tissue slurry, filter it through a 200-mesh nylon net into a test tube; centrifuge at 1000r/min for 5 minutes. Then wash three times with PBS, each time centrifuging at a low speed of 800r/min for 5 minutes to remove cell debris; filter out cell clumps with a 350-mesh nylon net, count the cells and adjust the cell concentration to 2×10⁶/ml.

2.6.2 Membrane Fixation

Divide the single cell suspension into two tubes (control tube and analysis tube), add 100μl of Intraprep Reagent I fixative to the prepared single cell suspension, shake vigorously, let stand at room temperature for 15 minutes, then add an appropriate amount of PBS to mix well, centrifuge at 1200r/min for 5 minutes at room temperature, repeat 2–3 times, discarding the supernatant. Then add 100μl of Intraprep Reagent II membrane agent to both tubes, shake well, and let stand at room temperature for 15 minutes.

2.6.3 Monoclonal Antibody Binding

Add 20μl of I antibody (1:50, NF-κB p65) to the analysis tube, and 20μl of PBS buffer to the control tube. Gently shake both tubes, incubate at room temperature for 30 minutes, then add an appropriate amount of PBS to both tubes, centrifuge at 1200r/min for 5 minutes at room temperature, repeat 2–3 times, discarding the supernatant, and simultaneously add 20μl of II antibody, incubate at room temperature for 30 minutes, allowing the reaction to proceed.

2.6.4 Sample Preparation

Add an appropriate amount of PBS to both tubes, shake well, centrifuge at 1200r/min for 5 minutes at room temperature, repeat 2–3 times, discarding the supernatant, then add 500μl of PBS containing 0.5% formaldehyde, preparing for machine testing [22–24].