Tecniche di biodosimetria: COMET ASSAY. Giorgia Aversa Mail: om

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1 Tecniche di biodosimetria: COMET ASSAY Giorgia Aversa Mail: om

2 The Comet Assay, also known as the Single Cell Gel Electrophoresis Assay, is a rapid, sensitive and relatively simple method that combines the simplicity of biochemical techniques for detecting DNA single and double strand breaks, alkali-labile sites, and cross-linking, with the single cell approach typical of cytogenetic assays. The comet assay can be conducted : Under neutral condition, in which the lysis and electrophoresis is made under neutral conditions. Under alkaline condition the unwinding and electrophoresis processes are made at ph 13 and this facilitates the detection of single and double strand breaks, expresses alkali labile sites (ALS) in addition to all types of lesions listed above. This method was developed to measure low levels of strand breaks with high sensitivity.

3 THE ALKALINE COMET ASSAY This technique provides 9 main points: 1) Preparation of slide with NMA 2) Deposition of the cells embedded in LMA on the slides 3) Cellular lysis to remove cellular membranes 4) Rising in electrophoretic buffer 5) Unwinding in electrophoretic buffer to unroll DNA strands 6) Electophoresis in basic condition, ph 13 7) Neutralization of alkaline products 8) Dehydratation with different alcoholic passages or in methanol 9) Staining of DNA and visualization of comet images

4 PREPARATION OF SLIDE The day before the experiment + mix choose the percentage of agarose (from 0.1% to 1%) and than PBS 0.01M Soak the microscope slides in this solution for few seconds Afterwards dry the slides and lay them in a stove at 50 C for 1 hour

5 DEPOSITION OF THE CELLS EMBEDDED IN LMA ON THE SLIDES 1 0.5% LMA + PBS 0.01M If mixture is prepared days earlier, melt in the microwave for a few seconds 2 The day of the experiment 20 µl of cells are mixed with 180 µl of 0.5% low melting agarose Whole blood can be directly mixed with LMA and dropped on slides. Cultured adherent cells this have need to be detached and after centrifuged and than 20 ul of pellet can be mixed with LMA and dropped on slides QuickTime e un decompressore sono necessari per visualizzare quest'immagine. 20µl of cells + 180µL of LMA Mix in a vial

6 After this preparation This mix must be positioned in the center of the slides prepared before and covered with coverslip Then the slides are let on ice for 10 minutes to solidify the gel During the procedure put some tinfoil sheets between the slides and the ice to avoid the wet of slides.

7 LYSIS This step serves to release the cells from the membrane Take off very gently the coverslip from the slides and position them in coplins containing 50ml of lysis buffer, for 1 hour at 4 C in the dark. The lysis buffer (ph 10) contains: in 200 ml H2O d 29,22 g NaCl (2,5 M) 7,44 g Na 2 EDTA (100 mm) 0,24g Tris-HCl (100 mm) 1,6 g NaOH ph needs to be adjusted using HCl and NaOH 1M. Shortly before use add 1% (volume) Triton X-100 and 10% DMSO, to prevent the damage caused by free radicals during the lysis.

8 RINSING During the rinsing process removes residue and alkaline salts For the rising process put the slides in coplins containing 50ml of electrophoresis buffer for 10 minutes at 4 C in the dark. The electrophoresis buffer (ph 13) contains: in 4 lt H2O d 1,48 g Na 2 EDTA (1 mm) 48 g NaOH After magnetic stirrer adjust ph using HCl and NaOH 1M.

9 UNWINDING With this step has the unwinding of the double helix of DNA Position the slides inside the electrophoretic chamber (the slides should be positioned so that the labels are on the side of the black electrode). Add a volume of electrophoretic buffer able to cover the slides with a layer of 0,5 cm (measure the volume of buffer utilized and use always the same amount). Let slides for 20 min at 4 C in the dark.

10 ELECTROPHORESIS During this step DNA migrates from anode to cathode and if fragmentation of DNA has occurred the fragments will migrate faster creating the comet tail. Slides, let in the same buffer, are submitted to electrophoresis, 30 min, 25V, 400mA (for a 20-slides chamber). Generally must be 2.6 V per cm of length of the electrophoretic chamber.

11 NEUTRALIZATION This step serves to neutralize the alkali present After the electrophoresis slides are washed in coplin jairs with neutralization buffer for 10 minutes. This buffer contains : Tris 0.4M (ph 7.5).

12 DEHYDRATATION This step is dry the gel on slides 5 minutes 5 minutes 5 minutes Etanhol 70% Etanhol 80% Etanhol 100% Slides are dehydratated with with different ethanol passages 70%, 80%, 100% (5 minutes each)

13 STAINING OF DNA AND VISUALIZATION OF COMETS IMAGES To stain DNA can be used several fluorochromes, in our lab we use ethidium bromide. The DNA of damaged cells appears like a comet with a head (nuclear region) and a tail. The tail is determined by fragments of DNA who migrate faster in the direction of the cathode while the undamaged DNA remains confined in the nucleus

14 The different types of migration have been classified in 5 categories that corresponds to 5 levels of genetic damages: Type 1: Comet without tail where the genetic material remain inside the nucleus: no genetic damage Type 2: Cell with a small tail, little migration of fragments of DNA: light genetic damage. Type 3: Cell with tail with evident migration due to a greater damage. Type 4: Cell with definite tail with a consistent amount of fragments Type 5: Almost all the DNA is present in the tail: severe genetic damage

15 THE DAMAGES CAN BE CALCULATE WITH DIFFERENT METHODS Visual Scoring For visual scoring about 200 cells for each slide are read with a fluorescence microscope and classified according to the 5 types of damage Index of damage is calculated by summing the product of the percent of the cells appertaining to each class (type of comet) for the correspondent number of class. ID= 0x(number cells of type 1) + 1x(number cells of type2) + 2x(number cells of type3) + 3x(number cells of type 4) + 4x (number cells of type 5).

16 Automated Score In image analysis system cells are observed and recorded by a fluorescent microscope provided with a camera associated to computer. The measurement of DNA migration can be performed using different soft-wares. With the softwares the analytic measures include tail area, % DNA of tail, tail moment, tail length, total tail intensity Tail length is the distance of DNA migration from the body of the nuclear core and it is used to evaluate the extent of DNA damage Tail moment is defined as the product of the tail length and the fraction of total DNA in the tail The % DNA of tail gives a clear indication of the appearance of the comets and, in addition, is linearly related to the DNA break frequency over a wide range of levels of damage

17 EXAMPLE OF RESULTS Alkaline Comet assay This graph is an example of results obtained with the comet assay. In this experiment was analyzed the genetic damage induced by ionizing radiation, 1h after radiation at different doses: 0; 0.1; 1; 5 Gy, with and without antioxidant treatment (ceria). In this case the slides were read by Visual Scoring A.U % DNA breaks 1 h after radiation wo/with Ce R 2 = 0,8614 R 2 = 0, Gy 0,1 Gy 1 Gy 5 Gy wo Ce with Ce Lineare (wo Ce) Lineare (with Ce)

18 THE COMET ASSAY-FPG This is a modified version of comet assay to detect oxidative damage to DNA by using enzyme formamido-pyrimidine glycosylase, Fpg The FPG specifically recognizes and submits to cleavage the oxidized purine bases, producing apurinic sites that are then converted to strand breaks by endonuclease activity of the enzyme

19 PROTOCOL OF COMET ASSAY-FPG The protocol of the Comet Assay remains unchanged There is only one additional step: THE INCUBATION WITH THE ENZYME FPG Immediately after lysis put the slides in coplins containing 1x flare buffer for 10 minutes to balance the ph to 8 (ph at which the enzyme works) 1x flare buffer contain: 10 ml 25x flare buffer 1 (by TREVIGEN, Fpg FLARE TM Assay Kit) 240 ml deionized water (milliq)

20 Incubate slides in a humidified chamber (eg racks boxes) for min with the enzyme, at 37 C. To create the "chamber" put absorbent paper soaked in distilled water in an empty box to create the right level of humidity for the proper functioning of the enzyme For each experimental point we should have slides treated with enzyme and slides treated with the buffer only. Control slides are covered with Flare reaction buffer only Fpg flare reaction buffer contains: 40 µl 25x flare buffer 110 µl 100x BSA Bring total volume to 1 ml with deionized water (milliq) - FPG + FPG Fpg slides are covered with the Flare buffer + Fpg (working solution) FPG working solution contains: 98 µl Fpg flare reaction buffer 2 µl diluited Fpg enzyme

21 EXAMPLE OF RESULTS Comet-Fpg assay The number of fragments produced by enzyme treatment is directly proportional to the oxidative damage to DNA, while the fragments sizes are inversely proportional to this. Consequently the suitable parameter to measure oxidative stress to DNA is the Tail Moment that considers both of the length and the intensity of the tail The oxidative damage can be calculated by comparing the avarage of the tail moment of the cells treated with enzyme and the avarage of the tail moment of the untreated cells

22 EXAMPLE OF RESULTS Comet-Fpg assay This is an example of result obtained with of Comet-Fpg assay. Damage induced by ionizing radiation is marked in yellow Damage induced by oxidative stress is marked in red Also in this case the red part of histogram represents the %DNA breaks induced by oxidative damage, inducted by ionizing radiation. But in this case cells were treated by an antioxidant, the quercitin

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