ApoSENSOR™ ADP/ATP Ratio Bioluminescent Assay Kit
Contents
1. 96 well plate and read the background luminescence Data A For higher accuracy let the reaction mix sit at room temperature to burn off low level ATP contamination for a few hours 4 For suspension cells transfer 10 l of the cultured cells 103 104 into luminometer plate 5 For adherent cells remove culture medium and treat cells 103 104 with 50 l of Nucleotide Releasing Buffer for 5 minutes at room temperature with gentle shaking Transfer into luminometer plate 6 After 2 minutes read the sample in a luminometer or luminescence capable plate reader Data B 7 To measure ADP levels in the cells read the samples from step 6 again Data C then add 1 l of ADP Converting Enzyme Read the samples after 2 minutes Data D Note The results can be analyzed using cuvette based luminometers or Beta Counters When Beta Counter is used it should be programmed in the out of coincidence or Luminescence mode for measurement The entire assay can directly be done in a 96 well plate It can also be programmed automatically using instrumentation with injectors When using injector the ATP Monitoring Enzyme and the ADP Converting Enzyme can be diluted with the Nuclear Releasing Buffer at 1 50 for injector The assay utilizes a glow type luciferase which has replaced the original flash type luciferase While still sensitive to sub picomole amounts of ATP the glow type reactions can still be re2. BioVision rev 02 13 BioVision Incorporated Tel 408 493 1800 Fax 408 493 1801 155 S Milpitas Boulevard Milpitas CA 95035 USA www biovision com tech biovision com Page 1 of 2 For research use only ApoSENSOR ADP ATP Ratio Bioluminescent Assay Kit Catalog K255 200 Store kit at 20 C I Introduction The changes in ADP ATP ratio have been used to differentiate the different modes of cell death and viability Increased levels of ATP and decreased levels of ADP have been recognized in proliferating cells In contrast decreased levels of ATP and increased levels of ADP are recognized in apoptotic cells The decrease in ATP and increase in ADP are much more pronounced in necrosis than apoptosis ApoSENSOR ADP ATP Ratio Assay kit utilizes bioluminescent detection of the ADP and ATP levels for a rapid screening of apoptosis necrosis growth arrest and cell proliferation simultaneously in mammalian cells The assay utilizes the enzyme luciferase to catalyze the formation of light from ATP and luciferin and the light can be measured using a luminometer or Beta Counter ADP level is measured by its conversion to ATP that is subsequently detected using the same reaction The assay can be fully automatic for high throughput and is highly sensitive can detect 100 mammalian cells well II Kit Contents III Reagent Reconstitution and General Consideration 1 Reconstitute ATP Monitoring Enzyme with 2 1 ml of the Enzy
3. ad an hour later This means that ATP amp ADP levels are now determined by quasi steady state light output levels This makes the reading of an entire 96 well 384 well plate much more feasible 8 Calculation ADP ATP Ratio is calculated as Data D Data C Data B Data A Interpretation of Results Cell Fate ADP Level ATP Level ADP ATP Proliferation Very Low High Very Low Growth Arrest Low Slightly Increased Low Apoptosis High Low High Necrosis Much Higher Very Low Much Higher The interpretation of different ratios obtained may vary significantly according to the cell types and conditions used However the following criteria may be used as guidelines a Test gives markedly elevated ATP values with no significant increase in ADP levels in comparison to control cells proliferation b Test gives similar or slightly higher levels of ATP and with little or no change in ADP compared to control growth arrest c Test gives lower levels of ATP to control but shows an increase in ADP apoptosis d Test gives considerable lower ATP levels than control but greatly increased ADP necrosis RELATED PRODUCTS ApoSENSORTM ATP Determination Kit Cell Viability Assay Kit K790 StayBrite Luciferase Luciferin Reagent K791 StayBrite ATP Assay Kit FOR RESEARCH USE ONLY Not to be used on humans Components K255 200 Cap Code Part Number Nucleotide Releasing Buffer ATP M
4. erve for lysis under microscope Lysates used after multiple free thaw cycles Aliquot and freeze sample lysates if needed to use multiple times Presence of interfering substance in the sample Troubleshoot if needed deproteinize samples Use of old or inappropriately stored samples Use fresh samples or store at correct temperatures till use Lower Higher readings in Samples Improperly thawed components Thaw all components completely and mix gently before use Use of expired kit or improperly stored reagents Always check the expiry date and store the components appropriately Allowing the reagents to sit for extended times on ice Always thaw and prepare fresh reaction mix before use Incorrect incubation times or temperatures Refer datasheet amp verify correct incubation times and temperatures Incorrect volumes used Use calibrated pipettes and aliquot correctly Unanticipated results Measured at incorrect wavelength Check the equipment and the filter setting Samples contain interfering substances Troubleshoot if it interferes with the kit Use of incompatible sample type Refer data sheet to check if sample is compatible with the kit or optimization is needed Sample readings above below the linear range Concentrate Dilute sample so as to be in the linear range Note The most probable list of causes is under each problem section Caus
5. es Solutions may overlap with other problems
6. me Reconstitution Buffer amp mix gently by inversion 2 Reconstitute ADP Converting Enzyme with 220 l of the Nucleotide Releasing Buffer amp mix gently by inversion 3 The reconstituted enzymes are stable for up to 2 months at 4 C after reconstitution For more accurate handling the reconstituted ADP Converting Enzyme can be diluted 10 fold with Nucleotide Releasing Buffer just before use Section IV Step 7 then use 10 l of the enzyme for each assay 4 ApoSENSOR is significantly more sensitive than other methods used for cell viability assays As a rule we recommend using 1 x 104 1 x 106 cells per assay Avoid contamination with ATP from exogeneous biological sources such as bacteria or fingerprints 5 Ensure that the Nucleotide Releasing Buffer is at room temperature before use The optimal temperature is 22 C Keep enzymes on ice during the assay and protect from light as much as possible 6 The assay can be performed using either a single tube or a white walled 96 well luminometer plate 100 l well culture volume is recommended IV ApoSENSOR ADP ATP Ratio Assay Protocol 1 Induce apoptosis in cells by desired method Concurrently incubate a control culture without induction 2 For each sample well to be measured mix 100 l of reaction mix consisting of ATP monitoring enzyme 10 l Nucleotide Releasing Buffer 90 l 3 Add 100 l of the reaction mix to the appropriate wells of a
7. onitoring Enzyme ADP Converting Enzyme Enzyme Reconstitution Buffer 50 ml Lyophilized Lyophilized 2 ml NM Green Blue Red K255 200 1 K255 200 2 K255 200 3 K255 200 4 BioVision rev 02 13 BioVision Incorporated Tel 408 493 1800 Fax 408 493 1801 155 S Milpitas Boulevard Milpitas CA 95035 USA www biovision com tech biovision com Page 2 of 2 For research use only GENERAL TROUBLESHOOTING GUIDE Problems Cause Solution Assay not working Use of a different buffer Refer data sheet and use buffers as indicated Omission of a step in the protocol Refer and follow the data sheet precisely Plate read at incorrect wavelength Check the wavelength in the data sheet and the filter settings of the instrument Use of a different 96 well plate Fluorescence Black plates Luminescence White plates Colorimeters Clear plates Samples with erratic readings Use of an incompatible sample type Refer data sheet for details about incompatible samples Samples prepared in a different buffer Use the Nucleotide releasing buffer provided in the kit or refer data sheet for instructions Samples were not deproteinized if indicated in datasheet Use the 10 kDa spin cut off filter or PCA precipitation as indicated Cell tissue samples were not completely homogenized Use Dounce homogenizer increase the number of strokes obs
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