Journal: Journal of Ovarian Research

Article Title: Sestrin1, 2, and 3 are dispensable for female fertility in mice

doi: 10.1186/s13048-024-01345-z

Figure Lengend Snippet: Construction of Sesn1 −/− , Sesn2 −/− , and Sesn3 −/− mice. ( A ) Limiting the expression of gene fragments for Sestrin1 , Sestrin2 , and Sestrin3 using the CRISPR/Cas9 system. ( B ) Genotyping of wild-type (WT) and knockout mice. ( C ) Detection of S esn1, Sesn2 and Sesn3 mRNA in Sesn1 −/− , Sesn2 −/− , and Sesn3 −/− and WT mice ovaries by Agarose gel electrophoresis of real time PCR. ( D ) Expression of SESN1, 2, and 3 in WT and knockout mice ovaries detected by IHC. Scale bar = 50 μm

Article Snippet: Ovarian tissue sections were deparaffinized, rehydrated, and subjected to antigen retrieval in 10 mM citrate buffer at 95 °C for 20 min. Primary antibodies, including Anti-Sestrin1 (Novus, NBP2-20316), Anti-Sestrin2 (Proteintech,10759-1-AP), and Anti-Sestrin3 (Proteintech,11431-2-AP), were applied at a 1:100 dilution and incubated overnight at 4 °C following the inhibition of endogenous peroxidase activity.

Techniques: Expressing, CRISPR, Knock-Out, Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction

Journal: Materials Today Bio

Article Title: Lactoferrin-cyanidin-3-glucoside nanoparticles alleviate inflammation and oxidative stress via Sesn2/Nrf2 activation in mastitis

doi: 10.1016/j.mtbio.2025.102491

Figure Lengend Snippet: Schematic illustration of lactoferrin-cyanidin-3-glucoside-encapsulated nanoparticles alleviating LTA-induced inflammation and oxidative stress via Sesn2/Nrf2 activation in mastitis.

Article Snippet: Following this, sections were blocked using goat serum to minimize non-specific binding and incubated overnight at 4 °C with a rabbit-derived primary antibody specific for SESN2 (Proteintech, 66297-1-Ig), Nrf2 (Proteintech, 16396-1-AP), and TNF-α (Abmart, PY19810) at a 1:300 dilution.

Techniques: Activation Assay

Journal: Materials Today Bio

Article Title: Lactoferrin-cyanidin-3-glucoside nanoparticles alleviate inflammation and oxidative stress via Sesn2/Nrf2 activation in mastitis

doi: 10.1016/j.mtbio.2025.102491

Figure Lengend Snippet: Effect of LF-C3GNPs on H 2 O 2 -induced oxidative stress and inflammation in HC11 cells via Sesn2/Nrf2 signaling activation. (A – D) Antioxidant enzyme activity of SOD, CAT, GPx and lipid peroxidation marker (MDA) levels in HC11 cells (n = 3). (E – L) RT-qPCR analysis (E – K) and Western blotting (L) of key redox regulators biomarkers (Nrf2, SLC7A11, Keap1, Sesn2, NQO1, HO-1, and GPX4) under different treatments (n = 3). (M – O) Immunofluorescence staining (M) and quantification (N, O) of Nrf2 and Sesn2 in HC11 (n = 3). Scale bar: 50 μm. Mean ± SD; # p<0.05 compared to the untreated control group; ∗ p<0.05 , ∗∗ p<0.01 , ∗∗∗ p<0.001 , and ∗∗∗∗ p<0.0001 relative to the H 2 O 2 -treated groups.

Article Snippet: Following this, sections were blocked using goat serum to minimize non-specific binding and incubated overnight at 4 °C with a rabbit-derived primary antibody specific for SESN2 (Proteintech, 66297-1-Ig), Nrf2 (Proteintech, 16396-1-AP), and TNF-α (Abmart, PY19810) at a 1:300 dilution.

Techniques: Activation Assay, Activity Assay, Marker, Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining, Control

Journal: Materials Today Bio

Article Title: Lactoferrin-cyanidin-3-glucoside nanoparticles alleviate inflammation and oxidative stress via Sesn2/Nrf2 activation in mastitis

doi: 10.1016/j.mtbio.2025.102491

Figure Lengend Snippet: Effect of LF-C3GNPs on S. aureus -induced oxidative stress and inflammation in mice via Sesn2/Nrf2 signaling activation . (A – D) Antioxidant enzyme activity of SOD, CAT, GPx and lipid peroxidation marker (MDA) levels in S. aureus -infected mammary tissue (n = 3). (E – L) Western blotting (E) and RT-qPCR analysis (F – L) of key redox regulators biomarkers (Nrf2, Sesn2, Keap1, SLC7A11, NQO1, HO-1, and GPX4) under different treatments (n = 3). (M – O) Immunofluorescence staining (M) and quantification (N, O) of Nrf2 and Sesn2 in S. aureus -infected mammary tissue (n = 3). Scale bar: 50 μm. Mean ± SD; # p<0.05 compared to the untreated control group; ∗ p<0.05 , ∗∗ p<0.01 , ∗∗∗ p<0.001 , and ∗∗∗∗ p<0.0001 relative to the S. aureus -treated groups.

Article Snippet: Following this, sections were blocked using goat serum to minimize non-specific binding and incubated overnight at 4 °C with a rabbit-derived primary antibody specific for SESN2 (Proteintech, 66297-1-Ig), Nrf2 (Proteintech, 16396-1-AP), and TNF-α (Abmart, PY19810) at a 1:300 dilution.

Techniques: Activation Assay, Activity Assay, Marker, Infection, Western Blot, Quantitative RT-PCR, Immunofluorescence, Staining, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Histone Demethylase JMJD3 Mediated Doxorubicin-Induced Cardiomyopathy by Suppressing SESN2 Expression

doi: 10.3389/fcell.2020.548605

Figure Lengend Snippet: The expression of JMJD3 and SESN2 in human dilated cardiomyopathy and DOX-induced chronic rat cardiomyopathy model. (A,B) The mRNA levels of JMJD3 and SESN2 were measured in the human dilated cardiomyopathy, n = 3 in Normal group, n = 7 in DCM group. SD rats were treated with DOX (accumulate dose 20 mg/kg) for 3 weeks or equal volume of normal saline (NS), n = 6 in NS group, n = 9 in DOX group. (C) The morphologic changes of gross hearts, scale bar = 5 mm. (D,E) Pathological changes of hearts tissues were detected by H&E staining (scale bar = 2 mm) and PSR staining (scale bar = 100 μm). (F) Echocardiographic graphs. (G,H) EF (%) and FS (%) were analyzed. (I,K) The mRNA expression of JMJD3 and SESN2 were measured in the rat hearts by qRT-PCR. (J,L) The protein expression of JMJD3 and SESN2 were detected by Western blot. (M) The protein level of SESN2 was measured in by IHC, scale bar = 100 μm. Data were presented as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 vs. Normal group or NS group. DCM, dilated cardiomyopathy; DOX, doxorubicin; EF, ejection fraction; FS, fractional shortening; HE, hematoxylin-eosin; i.p., intraperitoneally injection; IHC, immunohistochemistry; JMJD3, Jumonji domain-containing 3; NS, normal saline; PSR, Picro Sirius Red; SESN2, Sestrin2; SD, Sprague-Dawley.

Article Snippet: Anti-SESN2 antibody (66297-1-Ig, Boston, MA, United States) was from Cell Signaling Technology.

Techniques: Expressing, Saline, Staining, Quantitative RT-PCR, Western Blot, Injection, Immunohistochemistry

Journal: Frontiers in Cell and Developmental Biology

Article Title: Histone Demethylase JMJD3 Mediated Doxorubicin-Induced Cardiomyopathy by Suppressing SESN2 Expression

doi: 10.3389/fcell.2020.548605

Figure Lengend Snippet: The expression changes of JMJD3 and SESN2 with chronic DOX-induced cardiotoxicity in NRCMs. NRCMs were incubated with 0.5 μM DOX for indicated time points or with different concentrations of DOX (0.1, 0.5, and 1 μM) for 48 h. (A) Cell morphology of cardiomyocytes was observed by using a light microscopy, scale bar = 100 μm. (B) Hoechst-staining was used to observe chromatin condensation (indicated by arrows), scale bar = 100 μm. (C) Depolarization of mitochondrial membrane potential was detected by TMRE staining, scale bar = 100 μm. (D,E) The protein expression of cleaved caspase3 were measured by Western blot. (F) The mRNA levels of JMJD family were determined by RNA-Seq analysis. (G,H) The mRNA level of JMJD3 was measured by qRT-PCR. (I–K) The protein levels of JMJD3 and SESN2 were measured by Western blot. Data were presented as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 vs. Con group. n = 3. DOX, doxorubicin; NRCMs, primary-cultured of neonatal rat cardiomyocytes; TMRE, tetramethyl rhodamine methyl ester; JMJD, Jumonji domain-containing; JMJD3, Jumonji domain-containing 3; SESN2, Sestrin2.

Article Snippet: Anti-SESN2 antibody (66297-1-Ig, Boston, MA, United States) was from Cell Signaling Technology.

Techniques: Expressing, Incubation, Light Microscopy, Staining, Membrane, Western Blot, RNA Sequencing, Quantitative RT-PCR, Cell Culture

Journal: Frontiers in Cell and Developmental Biology

Article Title: Histone Demethylase JMJD3 Mediated Doxorubicin-Induced Cardiomyopathy by Suppressing SESN2 Expression

doi: 10.3389/fcell.2020.548605

Figure Lengend Snippet: JMJD3 overexpression aggravated DOX-induced cardiomyopathy in vitro and in vivo. NRCMs were infected with Ad-JMJD3 or Ad-GFP (empty vector) for 48 h. (A) Morphology of cardiomyocytes was observed by a light microscopy, scale bar = 50 μm. (B) Chromatin condensation was shown by Hoechst staining (indicated by arrows), scale bar = 50 μm. (C) The mitochondrial structure of cardiomyocytes was observed by mitotracker Red staining, scale bar = 25 μm. (D) Depolarization of mitochondria membrane potential was detected by TMRE-staining, scale bar = 100 μm. (E,F) The protein changes of BAX/Bcl-2 and cleaved caspase3 were analyzed by Western blot. Moreover, Ad-JMJD3 or Ad-GFP was transduced into the rat left ventricle via intramyocardial in situ injection. (G) The morphologic changes of gross hearts, scale bar = 5 mm. (H) Pathological changes of hearts tissues were detected by Masson-staining, scale bar = 100 μm. (I) Representative echocardiographic graphs. (J,K) EF (%) and FS (%) were analyzed by echocardiography, n = 8. The cardiac function was measured in 1, 2, and 3 weeks after the transfection of Ad-JMJD3 or Ad-GFP. (L) The protein expression of SESN2 was measured by Western blot. Data were presented as the mean ± SD. ** p < 0.01, *** p < 0.001 vs. Ad-GFP group. n = 3. DOX, doxorubicin; EF, ejection fraction; FS, fractional shortening; JMJD3, Jumonji domain-containing 3; SESN2, Sestrin2; NRCMs, primary-cultured of neonatal rat cardiomyocytes; TMRE, tetramethyl rhodamine methyl ester.

Article Snippet: Anti-SESN2 antibody (66297-1-Ig, Boston, MA, United States) was from Cell Signaling Technology.

Techniques: Over Expression, In Vitro, In Vivo, Infection, Plasmid Preparation, Light Microscopy, Staining, Membrane, Western Blot, In Situ, Injection, Transfection, Expressing, Cell Culture

Journal: Frontiers in Cell and Developmental Biology

Article Title: Histone Demethylase JMJD3 Mediated Doxorubicin-Induced Cardiomyopathy by Suppressing SESN2 Expression

doi: 10.3389/fcell.2020.548605

Figure Lengend Snippet: JMJD3 decreased H3K27me3 enrichment in the promoter region of SESN2. (A) Cardiomyocytes were co-treated with DOX (1 μM) and proteasome inhibitor MG132 (10 μM) for 12 h. (B) Cardiomyocytes were stimulated with DOX at relative lower concentration (0.5 μM) for longer time (48 h) and MG132 (10 μM) were co-treated. The protein expression of SESN2 were detected by western blot. (C,D) The mRNA and protein expression of SESN2 were measured by qRT-PCR and Western blot. Data were presented as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 vs. Con or Ad-GFP group. # p < 0.05, ## p < 0.01 vs. Ad-GFP + DOX group, ns means no significant differences. n = 3. ChIP assay was performed to detect JMJD3 and H3K27me3 enrichment on the SESN2 promoter region in NRCMs. (E) The immunoprecipitated protein-DNA complexes were detected with primers of SESN2. (F,G) The results of NRCMs treat with or without DOX (0.5 μM) for 48 h. (H,I) Results of cells treated with Ad-JMJD3 or Ad-GFP. Data were presented as the mean ± SD. *** p < 0.001 vs. Con group or Ad-GFP group. n = 3. DOX, doxorubicin; JMJD3, Jumonji domain-containing 3; SESN2, Sestrin2; ChIP, chromatin immunoprecipitation.

Article Snippet: Anti-SESN2 antibody (66297-1-Ig, Boston, MA, United States) was from Cell Signaling Technology.

Techniques: Concentration Assay, Expressing, Western Blot, Quantitative RT-PCR, Immunoprecipitation, Chromatin Immunoprecipitation

Journal: Frontiers in Cell and Developmental Biology

Article Title: Histone Demethylase JMJD3 Mediated Doxorubicin-Induced Cardiomyopathy by Suppressing SESN2 Expression

doi: 10.3389/fcell.2020.548605

Figure Lengend Snippet: Downregulation of SESN2 contributed to cardiotoxicity induced by JMJD3 overexpression. SESN2 was overexpressed by adenovirus Ad-SESN2 or knocked out by targeting sgRNA in NRCMs with or without DOX (0.5 μM, 48 h) and JMJN3 co-treatment. (A,E,H) The nuclear condensation in NRCMs was observed by Hoechst staining, scale bar = 100 or 50 μm. (B,F,I) Mitochondrial structure changes of cardiomyocytes was indicated by Mitotracker Red staining, scale bar = 25 μm. (C,D,G) The protein expression of SESN2, cleaved caspase3 and BAX/Bcl2 were analyzed by Western blot. Data were presented as the mean ± SD. (C,D) * p < 0.05, *** p < 0.001 vs. Ad-Flag or CRISPR-V2 group. ### p < 0.001 vs. Ad-Flag + DOX group. n = 3. (G) * p < 0.05 vs. Ad-GFP group. # p < 0.05 vs. Ad-GFP + DOX group. & p < 0.05 vs. Ad-JMJD3 + DOX group. n = 3. DOX, doxorubicin; JMJD3, Jumonji domain-containing 3; SESN2, Sestrin2.

Article Snippet: Anti-SESN2 antibody (66297-1-Ig, Boston, MA, United States) was from Cell Signaling Technology.

Techniques: Over Expression, Staining, Expressing, Western Blot, CRISPR

Journal: Frontiers in Cell and Developmental Biology

Article Title: Histone Demethylase JMJD3 Mediated Doxorubicin-Induced Cardiomyopathy by Suppressing SESN2 Expression

doi: 10.3389/fcell.2020.548605

Figure Lengend Snippet: Scheme of this study. Chronic DOX stimulation or JMJD3 overexpression in NRCMs induced cardiomyocytes apoptosis, which was relieved by JMJD3 knockdown or GSK-J4 (enzyme activity inhibitor of JMJD3). The increased expression of JMJD3 was negatively correlated with the decreased expression of SEESN2 following DOX stimulation. Mechanically, JMJD3 catalyzed the demethylation of H3K27me3 in the promoter region of SESN2. Subsequently, the transcription of SESN2 was decreased and mitochondrial function was disrupted, which ultimately induced cardiomyocytes apoptosis and cardiomyopathy.

Article Snippet: Anti-SESN2 antibody (66297-1-Ig, Boston, MA, United States) was from Cell Signaling Technology.

Techniques: Over Expression, Knockdown, Activity Assay, Expressing

Journal: bioRxiv

Article Title: Physical activity improves sarcopenia in a murine model by enhancing the proliferative potential of muscle stem cells, oxidative capacity of mitochondrial enzymes and expression of Sestrins

doi: 10.1101/811638

Figure Lengend Snippet: Quantification of Sestrin 1 and Sestrin 2: (a & b) Surface Plasmon Resonance and (c & d) western blot lower panel (n=3-4).

Article Snippet: A CM5 sensor chip (Wipro GE Healthcare, UK) was used to immobilize the antibodies via goat anti-mouse Sestrin1 IgG (SC-376170, Santa Cruz, USA) and rabbit anti-mouse Sestrin 2 IgG (SC-292558, Santa Cruz, USA).

Techniques: SPR Assay, Western Blot

Journal: Archives of biochemistry and biophysics

Article Title: Activation of Sestrin2 accelerates deep second-degree burn wound healing through PI3K/AKT pathway.

doi: 10.1016/j.abb.2023.109645

Figure Lengend Snippet: Fig. 1. SESN2 is upregulated during the deep second-degree burn wound healing process. (A) The healing process of deep second-degree burns in C57BL/6 mice. Relative (B) mRNA and (C) protein level of SESN2 in wound tissues collected at indicated healing time points. (D) Immunohistochemical analysis of SESN2 expression at murine wound edge tissues, scale bar: 100 μm. (E) SESN2 expression levels in uninjured and injured keratinocytes from GEO dataset (GSE30355). The data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, n = 5.

Article Snippet: After incubation with SESN2 antibody (1:500) overnight at 4 ◦C, the sections were incubated with secondary antibody horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (1: 1000, Wuhan Boster Biological Technology, Wuhan, China) at room temperature for 30 min.

Techniques: Immunohistochemical staining, Expressing

Journal: Archives of biochemistry and biophysics

Article Title: Activation of Sestrin2 accelerates deep second-degree burn wound healing through PI3K/AKT pathway.

doi: 10.1016/j.abb.2023.109645

Figure Lengend Snippet: Fig. 2. Up-regulation of SESN2 accelerates the burn wound healing process (A) Representative images and the (B) quantification of the burn wound area of mice in control group, eupatilin treated group and si-SESN2 group at indicated time (0 d, 7 d, 14 d, and 21 d). (C) HE and (D) Masson staining of tissue sections from different groups (control, eupatilin and si-SESN2), scale bar for HE: 1 mm and Masson: 400 μm. The data are presented as the mean ± SD. *P < 0.05, **P < 0.01, n = 5.

Article Snippet: After incubation with SESN2 antibody (1:500) overnight at 4 ◦C, the sections were incubated with secondary antibody horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (1: 1000, Wuhan Boster Biological Technology, Wuhan, China) at room temperature for 30 min.

Techniques: Control, Staining

Journal: Archives of biochemistry and biophysics

Article Title: Activation of Sestrin2 accelerates deep second-degree burn wound healing through PI3K/AKT pathway.

doi: 10.1016/j.abb.2023.109645

Figure Lengend Snippet: Fig. 3. SESN2 promotes the re-epithelialization during burn wound healing process. (A) The mRNA level of cytokines including TNF-α, IL-1β and IL-6 in murine wound edge tissues during the wound healing process. (B-C) The immunofluorescence staining of SESN2 (B), Ki67 (C) at murine wound edges of mice in control group, eupatilin treated group and si-SESN2 group, scale bar: 200 μm. The data are presented as the mean ± SD. **P < 0.01.

Article Snippet: After incubation with SESN2 antibody (1:500) overnight at 4 ◦C, the sections were incubated with secondary antibody horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (1: 1000, Wuhan Boster Biological Technology, Wuhan, China) at room temperature for 30 min.

Techniques: Immunofluorescence, Staining, Control

Journal: Archives of biochemistry and biophysics

Article Title: Activation of Sestrin2 accelerates deep second-degree burn wound healing through PI3K/AKT pathway.

doi: 10.1016/j.abb.2023.109645

Figure Lengend Snippet: Fig. 4. SESN2 promotes keratinocyte proliferation and migration. SESN2 expression levels in HaCaT cell treated with or without heat were measured by (A) qRT-PCR and (B) western blotting. The (C) mRNA and (D) protein levels of SESN2 in HaCaT cells treated with indicated concentrations of eupatilin. HaCaT cells pre-treated with heat were treated with or without eupatilin. (E) Cell viability was measured by CCK-8 assay, and (F) EDU assay was employed to assess the cell proliferation. (G–H) Wound healing assay (G) images and (H) quantitative analysis of HaCaT cells treated with or without 50 μM eupatilin at indicated time. (I) Transwell assay images and quantitative analysis of the migration of HaCaT cells treated with or without 50 μM eupatilin. The data are presented as the mean ± SD. *P < 0.05, **P < 0.01.

Article Snippet: After incubation with SESN2 antibody (1:500) overnight at 4 ◦C, the sections were incubated with secondary antibody horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (1: 1000, Wuhan Boster Biological Technology, Wuhan, China) at room temperature for 30 min.

Techniques: Migration, Expressing, Quantitative RT-PCR, Western Blot, CCK-8 Assay, EdU Assay, Wound Healing Assay, Transwell Assay

Journal: Archives of biochemistry and biophysics

Article Title: Activation of Sestrin2 accelerates deep second-degree burn wound healing through PI3K/AKT pathway.

doi: 10.1016/j.abb.2023.109645

Figure Lengend Snippet: Fig. 5. Deficiency of SESN2 impairs keratinocyte proliferation and migration. The knockdown efficiency of si-SESN2 in HaCaT cell pretreated with heat was confirmed by (A) qRT-PCR and (B) western blot assay. HaCaT cells pre-treated with heat were transfected with si-SESN2 or si-NC. (C) Cell viability was measured by CCK-8 assay, and (D) EDU assay was employed to evaluate the cell proliferation. (E–F) Wound healing assay (E) images and (F) quantitative analysis of HaCaT cells transfected with si-SESN2 or si-NC at indicated time. (G) Transwell assay images and quantitative analysis of the migration of HaCaT cells transfected with si-SESN2 or si-NC. The data are presented as the mean ± SD. *P < 0.05, **P < 0.01.

Article Snippet: After incubation with SESN2 antibody (1:500) overnight at 4 ◦C, the sections were incubated with secondary antibody horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (1: 1000, Wuhan Boster Biological Technology, Wuhan, China) at room temperature for 30 min.

Techniques: Migration, Knockdown, Quantitative RT-PCR, Western Blot, Transfection, CCK-8 Assay, EdU Assay, Wound Healing Assay, Transwell Assay

Journal: Archives of biochemistry and biophysics

Article Title: Activation of Sestrin2 accelerates deep second-degree burn wound healing through PI3K/AKT pathway.

doi: 10.1016/j.abb.2023.109645

Figure Lengend Snippet: Fig. 6. PI3K/AKT pathway mediates SESN2-deriven keratinocyte proliferation and migration. (A) Western blotting and quantitative analysis of SESN2, PI3K, p-PI3K, AKT, and p-AKT expression in HaCaT cells treated with eupatilin or LY294002. (B) Western blotting and quantitative analysis of SESN2, PI3K, p-PI3K, AKT, and p- AKT expression in HaCaT cells treated with 740Y-P with or without the knockdown of SESN2. (C) CCK-8 assay of HaCaT cells treated with eupatilin or LY294002 at indicated time. (D) Transwell assay images and quantitative analysis of HaCaT cells treated with eupatilin or LY294002. (E) CCK-8 assay of HaCaT cells treated with 740Y-P with or without the knockdown of SESN2 at indicated time. (F) Transwell assay images and quantitative analysis of HaCaT cells treated with 740Y-P with or without the knockdown of SESN2. The data are presented as the mean ± SD. *P < 0.05, **P < 0.01, NS, no significance.

Article Snippet: After incubation with SESN2 antibody (1:500) overnight at 4 ◦C, the sections were incubated with secondary antibody horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (1: 1000, Wuhan Boster Biological Technology, Wuhan, China) at room temperature for 30 min.

Techniques: Migration, Western Blot, Expressing, Knockdown, CCK-8 Assay, Transwell Assay