Dual Immunofluorescence of γH2AX and 53BP1 in Human Peripheral Lymphocytes

The aim of this manuscript was to evaluate the suitability of the simultaneous quantification of Gamma H2AX and 53BP1 foci to assess the genomic damage induced in human peripheral lymphocytes by bleomycin. Several factors can cause double strand breaks. Cells are thus provided with several repair mechanisms, involving both Gamma H2AX and 53 Binding Protein 1, that can co-localize near the double strand break.

After collecting whole blood samples, add 300 microliters of sample to a tube containing 4.7 milliliters of complete medium. Then add five micrograms per milliliter of bleomycin sulfate final concentration. For each sample, set up a negative control.

Put the tube in a thermostat at 37 degrees Celsius for two hours. Centrifuge samples at 540 G for five minutes. Then add five milliliters of hypotonic solution to pellet.

Then add 400 microliters of pre fixative solution in order to cause hemolysis. Centrifuge samples at 540 G for five minutes, then resuspend pellet in five milliliters of methanol at room temperature for at least 30 minutes to fix the cells. Centrifuge samples at 540 G for five minutes, then resuspend pellet in five milliliters of fixative solution.

Repeat this step once more time. Centrifuge again at 540 G for five minutes, then aspire the supernatant leaving enough solution to resuspend pellet. Drop the resuspended cell pellet on the slides.

Prepare the two solutions containing the anti-Gamma H2AX and the anti-53BP1 primary antibodies dissolved in blocking solution. Then wash slides two times in PBS 1X. Then keep slides for 30 minutes in blocking solution.

Add to each slide 10 microliters of each of the two solutions containing the primary antibodies dissolved in blocking solution. Cover the slides with parafilm. Incubate at four degrees Celsius overnight.

After the incubation, perform three washing in PBS 1X for five minutes. Prepare the two solutions containing the DyLight 488 and Alexa Fluor 568 secondary antibodies dissolved in blocking solution. Then add to each slide 10 microliters of each of the two solutions containing the secondary antibodies dissolved in blocking solution.

Cover the slides with parafilm and incubate at room temperature for two hours. After this second incubation, perform three washing in PBS 1X for five minutes. Add 2.5 microliters of Antifade solution with DAPI on the cover slips before assembly to counter stain the nuclei.

In order to assess foci kinetic, the procedure is the same as now described. The last phase is observation and quantification of foci. Human lymphocytes without Gamma H2AX and 53BP1 foci are shown in A, in B, and in C, human lymphocytes with different amounts of Gamma H2AX and 53BP1 foci are shown.

It is observed a low level of foci co-localization. As expected, a very higher frequency of both Gamma H2AX and 53BP1 foci are observed between untreated untreated cells. Also, a large difference is observed in the number of foci of the two markers.

The time course of Gamma H2AX and 53BP1 foci over time showed a different behavior although they contribute to the same function. Gamma H2AX and 53BP1 foci analysis has the ability to evaluate the cellular response in either exposure or pathological contexts. However, Gamma H2AX and 53BP1 dual immunofluorescence can lead to underestimate the true frequency of the event.