Understanding when and why cell cycle arrest occurs is critical to many fields of research, including (but not limited to) studies of development, aging, and cancer.
We all know the best tools produce the best results, so make sure you have all your bases covered with this list of the top 10 targets for your senescence research!
Number 1: β-Galactosidase Staining Kit
Senescent cells are known to express β-Galactosidase in a pH-dependent fashion, specifically detectable at pH 61. Our comprehensive Senescence β-Galactosidase Staining Kit #9860 contains everything you need to detect β-galactosidase activity at pH 6 in cells—or even frozen tissue. Perfect for quickly and easily testing multiple cell populations or tissue samples, the directions are straightforward, and the blue staining is bright and clear.
β-Galactosidase staining at pH 6 on normal WI38 cells at population doubling 29 (left) and senescent WI38 cells at population doubling 36 (right) using the Senescence β-Galactosidase Staining Kit #9860.
Number 2: p53 and phospho-53
p53 is so well-studied it, almost needs no introduction. A major player in the DNA Damage Response (DDR) pathways, the p53 tumor suppressor protein is also a critical regulator of the cell cycle, where accumulated phosphorylated p53 will drive activation of cyclin-dependent kinase inhibitors (CDKIs) and ultimately lead to cell cycle arrest.
Confocal Immunofluorescent analysis of HT-29 cells using p53 (7F5) Rabbit mAb (green). Actin filaments have been labeled with DY-554 phalloidin (red).
As mentioned above, senescent cell-cycle arrest relies heavily on phospho-53, which accumulates and activates multiple different CDKIs. Comparative analysis between p53 and phospho-p53 levels is often a critical step in studying the DDR pathway and senescence.
Confocal immunofluorescent analysis of MCF-7 cells, untreated (left) or etoposide-treated (right), using Phospho-p53 (Ser46) Antibody (green). Actin filaments have been labeled with DY-554 phalloidin (red).
Number 3: p21
One of the most well-established senescence markers, p21 is a CDKI downstream of phospho-p53. p21 acts as an inhibitor of the cell cycle by blocking progression through G1/S when associated with CDK2 (1).
Confocal immunofluorescent analysis of MCF7 cells using p21 Waf1/Cip1 (12D1) Rabbit mAb (red) and Phospho-Histone H3 (Ser10) (6G3) Mouse mAb #9706 (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
Number 4: p16
Another common and reliable senescence marker, expression of p16 is thought to drive cells into senescence2. p16 is a member of the INK4 family of CDKIs, which acts with CDK4 and CDK6 to arrest the cell cycle in G13.
Western blot analysis of extracts from HeLa and HUVEC cells using p16 INK4A (D3W8G) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
Number 5: LaminB1Senescent cells often exhibit morphological changes, making LaminB1 another useful indicator of senescence. A marker for nuclear morphology, LaminB1 expression is lost in senescent human and murine cells4. Loss of LaminB1 and increased accumulation of p21 and p16 are all important, classic hallmarks of senescence.
Confocal immunofluorescent analysis of HT-29 cells using Lamin B1 (119D5-F1) Mouse mAb (green) and β-Actin (13E5) Rabbit mAb (Alexa Fluor® 647 Conjugate) #8584 (red). Blue pseudocolor = Propidium Iodide (PI)/RNase Staining Solution #4087 (fluorescent DNA dye).
Number 6: Senescence-Associated Secretory Phenotype (SASP)
Senescent cells have many common traits, but they are not by any means identical. Each senescent population is characterized by unique levels of cytokines, growth factors, and proteases; this is called the senescence-associated secretory phenotype (SASP). The SASP Antibody Sampler Kit contains a robust collection of antibodies for various proteins for studying senescent cells. This collection will allow you to determine the SASP specific to your cell population.
Western blot analysis of recombinant Human Interleukin-1β (hIL-1β) #8900 using IL-1β (D3U3E) Rabbit mAb.
Number 7: Rb and phospho-Rb
Typically, phosphorylation of Rb is necessary to relieve repression of transcriptional targets and progress the cell cycle. Inhibition of the cell cycle by various CDKIs, including p21 and p16, leads to hyperactivation of Rb; this ultimately promotes the arrest of the cell cycle and senescence4.
Confocal immunofluorescent image of SH-SY5Y cells using RB (4H1) Mouse mAb (green). Actin filaments have been labeled with Alexa Fluor 555 phalloidin (red).
Since Rb must be phosphorylated to progress the cell cycle, phospho-Rb is not found in senescent cells. Like p53, comparative analysis of Rb and phospho-Rb is paramount when investigating senescence.
Confocal immunofluorescent analysis of MCF7 (left) and BT-549 (right) cells, untreated (upper) or λ phosphatase-treated (lower) using Phospho-Rb (Ser807/Ser811) (D20B12) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ® #4084 (fluorescent DNA dye).
Number 8: gamma-H2A.X
gamma-H2A.X is a classic marker of the DDR pathway. DNA damage results in a quick and robust response where H2A.X is phosphorylated at Ser139, forming gamma H2A.X (5), making it a powerful tool for studying the DDR pathway and senescence.
Immunohistochemical analysis of paraffin-embedded HT-29 cells untreated (left) or UV-treated (right), using Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb.
Number 9: 53BP1
53BP1 was originally identified as a binding partner for p53, and suggested to enhance its transcriptional activity6, 7. 53BP1 plays an essential role in DNA repair; it is known to be recruited to sites of DNA damage, and retention of 53BP1 at these sites is dependent on gamma-H2A.X8.
Confocal immunofluorescent analysis of HeLa cells using 53BP1 Antibody (green). Actin filaments have been labeled with Alexa Fluor® 555 phalloidin (red).
Number 10: Ki67
Sometimes the best way to detect something is to determine what it’s not doing. Ki67 is a nuclear protein that is a frequently used marker of proliferating cells. This detection ranges anywhere from G1 through the end of mitosis, but is not detectable when cells are in G0 resting phase9. A hallmark of senescent cells is a permanent exit from the cell cycle, and so senescent cells do not express Ki67.
Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Ki-67 (8D5) Mouse mAb.
Antibody Selection for Senescence Research
There are many markers of senescent cells, so why choose just one? We recommend starting with the Senescence Marker Antibody Sampler Kit, which contains several of these fundamental markers, making it perfect for beginning to identify your senescent cells.
You can also refer to our interactive DNA Damage Response (DDR) pathways to help identify relevant targets.
Select References:
- Hernandez-Segura A, Nehme J, Demaria M. Hallmarks of cellular senescence. Trends in Cell Biology. 2018;28(6):436-453. doi:10.1016/j.tcb.2018.02.001
- LaPak KM, Burd CE. The molecular balancing act of p16(INK4a) in cancer and aging. Mol Cancer Res. 2014;12(2):167-183. doi:10.1158/1541-7786.MCR-13-0350
- He S, Sharpless NE. Senescence in Health and Disease. Cell. 2017;169(6):1000-1011. doi:10.1016/j.cell.2017.05.015
- Gorgoulis V, Adams PD, Alimonti A, et al. Cellular Senescence: Defining a Path Forward. Cell. 2019;179(4):813-827. doi:10.1016/j.cell.2019.10.005
- Sharma A, Singh K, Almasan A. Histone H2AX phosphorylation: a marker for DNA damage. Methods Mol Biol. 2012;920:613-626. doi:10.1007/978-1-61779-998-3_40
- Iwabuchi K, Bartel PL, Li B, Marraccino R, Fields S. Two cellular proteins that bind to wild-type but not mutant p53. Proc Natl Acad Sci U S A. 1994;91(13):6098-6102. doi:10.1073/pnas.91.13.6098
- Ward IM, Minn K, Jorda KG, Chen J. Accumulation of checkpoint protein 53BP1 at DNA breaks involves its binding to phosphorylated histone H2AX. J Biol Chem. 2003;278(22):19579-19582. doi:10.1074/jbc.C300117200
- Wang B, Matsuoka S, Carpenter PB, Elledge SJ. 53BP1, a mediator of the DNA damage checkpoint. Science. 2002;298(5597):1435-1438. doi:10.1126/science.1076182
- Lawless C, Wang C, Jurk D, Merz A, Zglinicki Tvon, Passos JF. Quantitative assessment of markers for cell senescence. Experimental Gerontology. 2010;45(10):772-778. doi:10.1016/j.exger.2010.01.018
