C4-2B parental and HOXB13K13A mutant were stained with γH2AX-A488 (Biolegend, Clone 2F3; San Diego, CA, USA) antibody or γH2AX-APC conjugated antibody (Biolegend; Clone 2F3; Cat# 613415). After staining, cells were fixed with 1% v/v paraformaldehyde and permeabilized with 0.1% Triton-X-100 for 5 min and blocked with 1X PBS buffer containing 3% w/v protease-free BSA (incubation buffer). The cells were rinsed twice with the incubation buffer, followed by centrifugation and resuspended in 0.5 mL of 1X PBS, and acquired on LSR Canto and analyzed by the FlowJo software v10.10.
Clone 2f3
Manufactured by BioLegend
Sourced in United States
Clone 2F3 is a monoclonal antibody that recognizes a specific antigen. It can be used for various immunological applications.
Automatically generated - may contain errors
Lab products found in correlation
Dm6000b, by Leica (1 mentions)
Fw4000, by Leica (1 mentions)
H3k56ac, by Active Motif (1 mentions)
Aq132f, by Merck Group (1 mentions)
Propidium iodide, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647 conjugated antibody, by BioLegend (1 mentions)
Zombie aqua fixable viability kit, by BioLegend (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Etoposide, by Merck Group (1 mentions)
Cisplatin, by Abcam (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
4336 apc 050, by Bio-Techne (1 mentions)
Me 180, by American Type Culture Collection (1 mentions)
Anti gapdh antibody, by Novus Biologicals (1 mentions)
Olaparib, by Selleck Chemicals (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
6 protocols using clone 2f3
1
Analysis of DNA Damage by γH2AX
2
Comparative DNA Damage Response in MEFs and CHO Cells
3
Quantifying DNA Damage and Apoptosis
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DNA double-strand breaks and apoptosis were detected by immunofluorescence using antibodies against phosphorylated histone H2AX at serine 139 (clone 2F3, BioLegend), and anti-53BP1 (A300-272A, BioLegend). As phosphorylated histone H2AX and 53BP1 form discrete foci at the sites of DNA double-strand breaks, DNA damage induction was evaluated by detecting foci. In cells undergoing apoptosis, DNA is fragmented; therefore, multiple DNA double-strand breaks are induced, which are detectable by homogeneous nuclear staining with an antibody against phosphorylated histone H2AX at serine 139, not with 53BP1 antibody. The primary antibodies were detected by Alexa Fluor 555-labed anti-mouse IgG (A21422, Thermo Fisher Scientific). Nuclei were counterstained with 1 μg/mL of DAPI. Images were captured with a fluorescence microscope (DM6000B, Leica) and analyzed using FW4000 software (Leica). The percentage of apoptotic cells was calculated by dividing the number of cells with heavily phosphorylated histone H2AX signals by the number of total cells determined by the number of DAPI-positive nuclei. The percentage of cells with DNA double strand breaks was calculated by dividing the number of cells with 53BP1 foci by the number of total cells determined by the number of DAPI-positive nuclei.
4
Immunofluorescence Analysis of Cellular Markers
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The cells (5 mL) were fixed with 20 μL of Schaudinn’s fixative (saturated HgCl2: ethanol, 2:1). Then 10 μL of fixed cells were uniformly spread on a poly-L-lysine-coated coverslip. The cells were washed using PBST (0.05% Triton X-100) for 10 min. The cells were blocked using a blocking solution (3% BSA, 10% normal goat serum, and 0.05% Triton X-100 in PBS) for 1 h at room temperature (RT). They were then incubated overnight with HA antibody (1:500 dilution, #3724S, CST, Danvers, MA, USA), γH2AX (1:200, Clone 2F3, BioLegend, USA), and H3K56ac (1:500 dilution; AB_2661786, Active Motif, Carlsbad, CA, USA) at 4 ℃ overnight. The samples were washed three times with PBST (0.05% Triton X-100) and incubated with FITC-conjugated anti-rabbit IgG antibody (1:1000, AQ132F, Millipore, Billerica, MA, USA) or TRITC-conjugated anti-rabbit IgG antibody (dilution ratio of 1:500, AP192R, Millipore, Billerica, MA, USA) for one hour at RT. The samples were stained with 1 μg/mL DAPI for 15 min and observed using a Delta Vision Elite deconvolution microscope system (Applied Precision/GE Healthcare, Boston, Massachusetts, USA).
5
Immunostaining of CENP-A and γ-H2A.X
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Aliquots (5 ml) of cell suspension were transferred to a centrifuge tube and 20 μl partial Schaudinn’s fixative (2:1 ratio of saturated HgCl2,: absolute ethanol) were added. After 5 min, cells were washed twice with methanol and then resuspended in 50 μl methanol; drops of this suspension were applied to a slide and air-dried. Immunostaining of Cna1, a CENP-A homolog, used a rabbit polyclonal antiserum (1:200 dilution) [a gift from Harmit Malik, see (26 (link),37 (link))] and γ-H2A.X immunostaining used a mouse monoclonal antibody (1:200 dilution; clone 2F3, BioLegend, San Diego, CA, USA). Both were detected with a fluorescence-labeled secondary antibody.
6
Cervical Cancer Cell Line Protocols
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Human cervical cancer cell lines SiHa and ME180 were obtained from ATCC and maintained in DMEM (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 5% humidified CO2. PARP inhibitor Olaparib (AZD2281) was purchased from Selleckchem, Houston, USA. Cisplatin was purchased from Bio Vision, CA, USA. Anti-PARP antibody [clone 46D11, #9532 (WB 1:1000 and IF 1:800)], anti-vimentin [clone D21H3, #5741 (WB 1:1000)] and cell lysis buffer were obtained from Cell Signaling Technology, MA, USA. Anti-PAR antibody [#4336-APC-050 (WB 1:1200 and IF 1:400)] was purchased from Trevigen, MD, USA. Anti-γH2A.X (p-Ser139) antibody [clone EP854(2)Y, #ab81299 (IF 1:2000)] was purchased from Abcam, Alexa flour 488 anti-γH2A.X (phospho-S139) [clone 2F3, #613405 (IF 1:400 and FACS 1:200)] antibody was purchased from Bio Legend, San Diego, California and anti-GAPDH antibody [clone 2D4A7, #NB300–328 (WB 1:3000)] was obtained from Imgenex. XRCC4 (IF 1:200) and RPA (IF 1:400) antibodies were gifted by Dr. A.S. Balajee, Columbia University, New York, USA. XRCC1 (IF 1:500), 53BP (IF 1:500), RAD51 (IF 1:100) and Ku80 (IF 1:400) antibodies were kind gift from Dr. Sathees C. Raghavan, Indian Institute of Science, Bangalore.
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