Bioluminescence Cytotoxicity Assay Kit

(Catalog #K312-500; 500 assays; Store at –20^oC)

I. Introduction:

Cell death or cytotoxicity is classically evaluated by the quantification of plasma membrane damage. Adenylate kinase (AK) is a ubiquitous protein present in all eukaryotic and prokaryotic cells and rapidly release into culture medium upon damage of the plasma membrane. The Bioluminescence Cytotoxicity Assay Kit is based on the measurement of AK in a simple one-step procedure involving two chemical reactions. The first reaction is the conversion of ADP to ATP by adenylate kinase that was released from the damaged cells. The second reaction utilizes luciferase to catalyze the formation of light from ATP and luciferin, and the light can be measured using a luminometer or beta counter. The assay is highly sensitive and can be fully automatic for high throughput.

1. 
   

   Kit Contents:

   
   

 

 

 

2. 
   

   Preparation of Reagents and General Considerations:

   
   

Reconstitute each vial of the AK Detection Reagent with 10 ml AK Assay Buffer. Mix gently. Allow the mixture to equilibrate for 15 min at room temperature before use. Use the reconstituted reagent within 6 hours, or 24 hours if stored at 4^oC. Unused reagent can be stored at –20^oC for up to 2 months. Once thawed, the reagent must not be refrozen. Ensure that all reagents are at room temperature before use. The optimal temperature is 22^oC.

3. 
   

   Assay Protocol:

   
   

0. 
   

   Treat cells by desired method. Concurrently incubate a control culture without treatment

   
   


1. 
   

   Transfer 100 μl of the culture medium into a 96 well plate.

   
   


2. 
   

   Add 100 μl of the reconstituted AK reagent to each well. Incubate for 5 minutes.

   
   

Note: If using 384 well plate, we recommend adding 20 μl of the culture medium and 30 μl of the AK Detection Reagent.

3. 
   

   Read in a Microplate Luminometer.

   
   

Note: Samples should be read within 30 minutes following the addition of the AK Detection Reagent. The reaction time should be kept consistent for all samples.

4. 
   

   Microplate Luminometers with Injectors:

   
   

If using a microplate luminometer equipped with reagent dispensers, the Dispenser should be primed with the AK Detection Reagent and set to dispense 100 μl (96well plate) or 30 μl (for 384-well plate). It is recommended a delay time of at least 5 minutes prior to measurement (but no more than 30 minutes) be incorporated after injection of the AK Detection Reagent. 1 second integrated reading is recommend.

 

FOR RESEARCH USE ONLY! Not to be used in human.