Alexa fluor 647 conjugated antibody

MEFs were generated from
B10^k.Rag1^−/− and
NOD^k.Rag1^−/−mice and
were treated with 5 μM etoposide (Sigma-Aldrich) for 1 h. MEFs were
allowed to recover for 15 h before fixation and staining with Alexa Fluor
647–conjugated antibody to phosphorylated H2A.X (Ser139; clone 2F3,
Biolegend) for flow cytometry.
CHO cells and Xrcc4^−/− CHO
cells were a kind gift from P. Jeggo (University of Sussex). Xrcc4 expression
vectors were constructed with the B10 or NOD allele of Xrcc4(or GFP control), preceded by a chicken β-actin intron
and followed by a sequence encoding GFP linked to the Xrcc4 C terminus via a T2A
peptide, under the control of the chicken β-actin promoter and CMV
enhancer. Xrcc4^−/− CHO cells were
transfected using Lipofectamine 3000 (Invitrogen), followed by treatment with 5
μM etoposide (Sigma-Aldrich) for 1 h, and cells were allowed to recover
for 15 h. Cell sorting was performed for GFP⁺ CHO cells on a
FACS Aria II (Becton Dickinson). GFP⁺ cells were stained with
the Zombie Aqua Fixable Viability kit (Biolegend) before fixation and staining
with Alexa Fluor 647–conjugated antibody to phosphorylated H2A.X
(Ser139; clone 2F3, Biolegend) and propidium iodide (eBioscience) for flow
cytometry analysis.

Human platelet activation was determined in heparinized whole blood drawn from the retro-orbital plexus of humanized platelet mice. Briefly, α_IIbβ₃ activation was detected using an AlexaFluor647-conjugated antibody (Clone: PAC1, Biolegend) while an AlexaFluor647-conjugated polyclonal antibody against human p-selectin (a generous gift from Rodger McEver) was used for quantification of α-granule secretion. Humanized platelet mouse blood was stimulated or not with 30 μM adenosine 5′-diphosphate (ADP; Sigma-Aldrich) or 5 μM PAR1-specific activating peptide (PAR1-AP), SFLLRN-NH₂ (Sigma-Aldrich), for 10 minutes in the presence of 2 mM CaCl₂ before analysis by flow cytometry.