cck8 detection reagent  (Beyotime)

Journal: Journal of Cellular and Molecular Medicine

Article Title: Alpha‐Fetoprotein Stimulates Cleavage of Membranal MICA /B on Liver Cancer Cell Lead to Escape Immune Surveillance of Natural Killer Cells

doi: 10.1111/jcmm.71076

Figure Lengend Snippet: The effect of AFP on the expression of MMP9 and MICA/B shedding on the membrane of HCC cells. (A) Western blotting experiment was performed to detect the expression levels of MMP9; (B) Grey scale values of MMP9 expression were calculated using Image J software and then statistically analysed and plotted; (C) qRT‐PCR experiment was used to check MMP9 mRNA expression levels; (D) Western blotting was performed to detect the expression levels of sMICA/B in HLE, HLE‐NC, HLE‐AFP cells; (E) Western blotting was performed to detect the expression levels of sMICA/B in HuH‐7, HuH‐7‐NC, HuH‐7‐shAFP cells; (F) ELISA were performed to detect the content of sMICA/B in HCC cell supernatants; (G) CCK‐8 experiments were performed to detect the IC50 of GM6001 and TAPI‐1; (H) Detection of MICA/B expression in HCC cells while treated with GM6001 and TAPI‐1 by flow cytometry; (I) Prism GraphPad(10.0) for statistical analysis and graphing of flow cytometry results; (J) Detection of sMICA/B in HCC cells supernatants while treated with GM6001 and TAPI‐1 by ELISA; (K) Western blotting experiment was performed to detect the expression levels of PI3K, AKT, p‐AKT and MMP9; (L) Grey scale values of protein expression were calculated using Image J software and then statistically analysed and plotted. ns, p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001. The picture represents at least three repetitions of the experiment. AKT, protein kinase B; GM6001 and TAPI‐1, MMP9 inhibitors; MMP9, matrix metallopeptidase 9; p‐AKT, phospho‐protein kinase B; PI3K, phosphatidylinositol‐3‐kinase; sMICA/B, soluble major histocompatibility complex class I chain‐related proteins A and B.

Article Snippet: The cells were cultured in an incubator for 24 h, after which the medium was discarded, fresh serum‐free medium (100 μL per well) was added, and CCK8 detection reagent (Cat #CK04, DOJINDO, Japan) was added (10 μL per well).

Techniques: Expressing, Membrane, Western Blot, Software, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, CCK-8 Assay, Flow Cytometry, Immunopeptidomics

Journal: Experimental Biology and Medicine

Article Title: miR-539-5p targets BMP2 to regulate Treg activation in B-cell acute lymphoblastic leukemia through TGF-β/Smads/MAPK

doi: 10.3389/ebm.2024.10111

Figure Lengend Snippet: Effects of miR-539-5p downstream gene BMP2 on proliferation and apoptosis of Nalm-6 cells. (A) After transfection with BMP2-siRNA or pcDNA-BMP2, the protein expression of BMP2 was detected by WB to verify the transfection efficiency. (B) CCK8 was used to detect the viability of Nalm-6 cells after intervention or overexpression of BMP2. (C) The apoptosis of Nalm-6 cells was detected by flow cytometry. (D) Histogram of percentage of apoptotic cells. (E) The protein expressions of Bax, Bcl-2 and PCAN were detected by WB. (F) Histogram of gray level analysis of protein bands. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: After different treatments, Nalm-6 cells were incubated with 10 μL CCK8 detection reagent (cat. no. C0037; Beyotime, China) for 1 h. The absorbance was measured at 450 nm and the cell viability was calculated.

Techniques: Transfection, Expressing, Over Expression, Flow Cytometry

Journal: Experimental Biology and Medicine

Article Title: miR-539-5p targets BMP2 to regulate Treg activation in B-cell acute lymphoblastic leukemia through TGF-β/Smads/MAPK

doi: 10.3389/ebm.2024.10111

Figure Lengend Snippet: Effects of miR-539-5p/BMP2 regulatory axis on proliferation and apoptosis of Nalm-6 cells. After transfection of Nalm-6 cells with miR-539-5p-mimic and/or pcDNA-BMP2, the gene expression of miR-539-5p (A) and BMP2 (B) was detected by RT-PCR. (C) The protein expression of BMP2 was detected by WB. (D) CCK8 was used to detect the viability of Nalm-6 cells. (E) The apoptosis of Nalm-6 cells was detected by flow cytometry. (F) Histogram of percentage of apoptotic cells. (G) The protein expressions of Bax, Bcl-2 and PCAN were detected by WB. (H) Histogram of gray level analysis of protein bands. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: After different treatments, Nalm-6 cells were incubated with 10 μL CCK8 detection reagent (cat. no. C0037; Beyotime, China) for 1 h. The absorbance was measured at 450 nm and the cell viability was calculated.

Techniques: Transfection, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Expressing, Flow Cytometry