Day 1, Prepare CHO cells for transfection

Count and seed same amounts of CHO cells into a 24-well plate (do not use 96-well plate). Make sure cell confluence is ~ 40%. There should be at least three wells of cells (three technical replicates) for each experimental groups. Do not use CHO cells which have been passaged for more than 20 generations. CHO culturing medium: 10% FBS, 5% Glutamax, 0.5% penicillin and streptomycin in F12 medium. 0.5 ml per well.

Day 2, Transfect CHO cells with HilyMax

(1) Make sure the plasmids were prepared using an endotoxin-free kit. The 260/280 value should be around 1.8. Adjust the concentration of all plasmids to 1 μg/μl.

(2) Check the cell confluence. After 24 hours, it should be ~70%.

(3) For each well, we used 1 μg plasmid and 5 μl HilyMax. Add 1 μg plasmid into 30 μl Opti-MEM (serum-free) and mix it well. Add 5 μl HilyMax into another 30 μl Opti-MEM and mix it well. Incubate both mixtures in dark at room temperature (RT) for 15 min. Then combine two mixtures together, vortex it well and incubate this final transfection mixture in dark at RT for 15 min.

(4) Replace the medium in 24-well plate to serum-free one (5% Glutamax, 0.5% penicillin and streptomycin in F12 medium). 0.5 ml per well. Then add the final transfection mixture. Four hours later, remove the transfection medium and add 0.5 ml culturing medium into each well.

Day 3, Induce acidosis

(1) Key step, 20 hour after transfection, validate the transfection rate for each well by checking GFP signal. We usually got ~80% transfection rate with this protocol. Make sure all wells have a similar transfection rate. Wells with distinct transfection rate cannot use to compare. Any well with a transfection rate lower than 50% cannot be used for detecting acidosis-induced cell death.

(2) Remove culturing medium in all wells and wash them twice with warm pH 7.4 treatment solution (150 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, and 10 mM glucose, buffered to pH 7.4 with 10 mM HEPES). For non-acidosis control, wash one more time with warm pH 7.4 treatment solution. Then add 0.5 ml of pH 7.4 treatment solution and incubate at 37°C for one hour. For acidosis group, wash one more time with warm pH 6.0 treatment solution. Then add 0.5 ml of pH 6.0 treatment solution (150 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, and 10 mM glucose, buffered to pH 6.0 with 10 mM HEPES) and incubate at 37°C for one hour.

(3) Use Cell Titer Blue to examine cell viability.