Mammalian HEK293T T cells stably expressing sr39-HSV1-TK (HEK293T-sr39TK) 1.
{"action": "clarify", "output": "", "device": [""]}
Incubate at 37 °C with 5% CO2 for 24 h. 2.
{"action": "incubate", "output": "", "temperature": ["37 \u00b0C"], "concentration": ["5% CO2"], "time": ["24 h"]}
Incubate at 37 °C for 3 h. Maintain the incubation time consistent between samples. #####%%%%% output "no HSV1-TK" are used in line8
{"action": "maintain", "output": "no HSV1-TK", "temperature": ["37 \u00b0C"], "time": ["3 h"]}
Keep the same cells (HEK293T) plated in equal numbers expressing no HSV1-TK as control. #####%%%%% output "control" are used in line12
{"action": "keep", "output": "control", "reagent": ["no HSV1-TK"], "temperature": [""]}
After 3 hours of incubation, remove the medium carefully without disturbing the cells, .
{"action": "remove", "output": "", "container": ["the medium"]}
wash the plates twice with 0.5 mL each of ice-cold PBS
{"action": "rinse", "output": "", "temperature": ["ice-cold"], "time": [""], "reagent": ["PBS"]}
Pool the medium solutions from respective wells to measure the remaining total activities. #####%%%%% output "the remaining total activities" are not used
{"action": "drain", "output": "the remaining total activities", "time": [""]}
Add 0.5 mL of 0.1 N NaOH to each well .
{"action": "add", "output": "", "reagent": ["0.1 N NaOH"], "container": ["each well"]}
leave it for 10 min at room temperature in a shaker to lyse the cells #####%%%%% output "cell lysate" are used in line20
{"action": "leave", "output": "cell lysate", "temperature": ["room temperature"]}
In brief, to each 20 μL of cell lysate add 1 mL of 1x Bradford protein assay reagent .
{"action": "add", "output": "", "reagent": ["cell lysate", "1x Bradford protein assay reagent"], "volume": ["20 μL", "1 mL"]}
read the absorbance at 595 nm (A595) 5 min after incubation at room temperature in a spectrophotometer #####%%%%% output "the absorbance" are not used
{"action": "read", "output": "the absorbance", "device": ["a spectrophotometer"], "length": [""]}
Use the standard graph prepared with the standards (BSA) diluted in 0.1 N NaOH to calculate the total protein present in 0.5 mL of cell lysate.
{"action": "use", "output": "", "reagent": ["cell lysate"]}
Transfer the remaining cell lysate (480 μL) to a scintillation vial . #####%%%%% output "3H-Penciclovir" are used in line28
{"action": "transfer", "output": "3H-Penciclovir", "volume": ["the remaining cell lysate (480 \u03bcL)"], "container": ["a scintillation vial"]}
Cells lacking HSV1-TK expression exposed to 3H-Penciclovir serve as a negative control. #####%%%%% output "a negative control" are not used
{"action": "provide", "output": "a negative control", "reagent": ["3H-Penciclovir"]}
Measure the total remaining activity from each sample by using 480 μL of solution taken from the pooled medium , . #####%%%%% output "3H-Penciclovir" are used in line32
{"action": "passage", "output": "3H-Penciclovir", "reagent": ["each sample"], "container": ["the pooled medium"], "length": [""]}
also measured from 0.5 μL of 3H-Penciclovir diluted in 480 μL of medium #####%%%%% output "different samples" are used in line34
{"action": "evaluate", "output": "different samples", "reagent": ["3H-Penciclovir"]}
Measure the radioactivity from different samples in a scintillation counter by taking an average reading/min by reading for 5 min. #####%%%%% output "an average reading/min" are not used
{"action": "passage", "output": "an average reading/min", "reagent": ["different samples"]}
Normalize the results with protein concentration . #####%%%%% output "3H-PCV" are used in line40
{"action": "normalize", "output": "3H-PCV", "concentration": ["protein concentration"]}
relate to the total activity

Results should be expressed as the percentage conversion of 3H-PCV per milligram of protein/total count, #####%%%%% output "3H-PCV" are used in line42
{"action": "confirm", "output": "3H-PCV", "reagent": ["3H-PCV"]}
as the conversion of 3H-PCV in moles per milligram of protein/total activity (Radioactivity within cells/[Radioactivity remained in medium Radioactivity within cells]). #####%%%%% output "protein/total" are not used
{"action": "keep", "output": "protein/total", "reagent": ["3H-PCV"], "temperature": [""]}