50-5699-82

antibody from Invitrogen Antibodies

Targeting: MKI67 MIB-1, PPP1R105

Immunocytochemistry (Supportive data in Antibodypedia)

Immunohistochemistry (Supportive data in Antibodypedia)

Flow cytometry (Supportive data in Antibodypedia)

Other assay (Supportive data in Antibodypedia)

Antibody Data

Product number

50-5699-82

Provider

Invitrogen Antibodies

Product name

Ki-67 Monoclonal Antibody (20Raj1), eFluor™ 660, eBioscience™

Antibody type

Monoclonal

Antigen

Other

Description

Description: The monoclonal antibody 20Raj1 recognizes the human Ki-67 protein. Two isoforms of Ki-67 exist, a 345 and 395 kDa form that are expressed in dividing cells. Ki-67 is expressed in all cell types and is detectable during active phases of the cell cycle (G1, S, G2, and mitosis) but is absent from resting cells (G0). During interphase, Ki-67 expression is localized to the nucleus but redistributes to the chromosomes during mitosis and has specifically been found to associate with heterochromatin-bound proteins such as chromobox protein homolog 3 (CBX3). In studies of tumor cells, Ki-67 expression has been used as a marker for determining the fraction of proliferating cells within a given population of tumor cells. This monoclonal antibody 20Raj1 recognizes canine Ki-67. Applications Reported: This 20Raj1 antibody has been reported for use in intracellular staining followed by flow cytometric analysis, microscopy, immunohistochemical staining, and immunocytochemistry. Applications Tested: This 20Raj1 antibody has been tested by immunocytochemistry on formaldehyde-fixed and permeabilized HeLa cells, immunohistochemistry on FFPE human skin using low pH antigen retrieval, and flow cytometry on stimulated normal human peripheral blood cells using the Foxp3/Transcription Factor Staining Buffer Set (Product # 00-5523-00) and protocol. For immunocytochemistry immunohistochemistry, this can be used at less than or equal to 20 µg/mL. For flow cytometry, this antibody can be used at less than or equal to 0.125 µg/test. A test is defined as the amount (µg) of antibody that will stain a cell sample in a final volume of 100 µL. Cell number should be determined empirically but can range from 10^5 to 10^8 cells/test. It is recommended that the antibody be carefully titrated for optimal performance in the assay of interest. eFluor® 660 is a replacement for Alexa Fluor® 647. eFluor® 660 emits at 659 nm and is excited with the red laser (633 nm). Please make sure that your instrument is capable of detecting this fluorochome. Excitation: 633-647 nm; Emission: 668 nm; Laser: Red Laser. Filtration: 0.2 µm post-manufacturing filtered.

Reactivity

Human, Canine

Host

Mouse

Isotype

IgG

Antibody clone number

20Raj1

Vial size

100 µg

Concentration

0.2 mg/mL

Storage

4° C, store in dark, DO NOT FREEZE!

References

Submitted references

Protein/AS01(B) vaccination elicits stronger, more Th2-skewed antigen-specific human T follicular helper cell responses than heterologous viral vectors.

Nielsen CM, Ogbe A, Pedroza-Pacheco I, Doeleman SE, Chen Y, Silk SE, Barrett JR, Elias SC, Miura K, Diouf A, Bardelli M, Dabbs RA, Barfod L, Long CA, Haynes BF, Payne RO, Minassian AM, Bradley T, Draper SJ, Borrow P
Cell reports. Medicine 2021 Mar 16;2(3):100207

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Functional Study of the Retrotransposon-Derived Human PEG10 Protease.

Golda M, Mótyán JA, Mahdi M, Tőzsér J
International journal of molecular sciences 2020 Mar 31;21(7)

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Single residue in CD28-costimulated CAR-T cells limits long-term persistence and antitumor durability.

Guedan S, Madar A, Casado-Medrano V, Shaw C, Wing A, Liu F, Young RM, June CH, Posey AD Jr
The Journal of clinical investigation 2020 Jun 1;130(6):3087-3097

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Human Liver Memory CD8(+) T Cells Use Autophagy for Tissue Residence.

Swadling L, Pallett LJ, Diniz MO, Baker JM, Amin OE, Stegmann KA, Burton AR, Schmidt NM, Jeffery-Smith A, Zakeri N, Suveizdyte K, Froghi F, Fusai G, Rosenberg WM, Davidson BR, Schurich A, Simon AK, Maini MK
Cell reports 2020 Jan 21;30(3):687-698.e6

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Streptomyces Levis ABRIINW111 Inhibits SW480 Cells Growth by Apoptosis Induction.

Faramarzian Azimi Maragheh B, Fatourachi P, Mohammadi SM, Valipour B, Behtari M, Dehnad A, Nozad Charoudeh H
Advanced pharmaceutical bulletin 2018 Nov;8(4):675-682

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Involvement of TWEAK and the NF-κB signaling pathway in lupus nephritis.

Sun F, Teng J, Yu P, Li W, Chang J, Xu H
Experimental and therapeutic medicine 2018 Mar;15(3):2611-2619

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Small-Vessel Vasculopathy Due to Aberrant Autophagy in LAMP-2 Deficiency.

Nguyen HT, Noguchi S, Sugie K, Matsuo Y, Nguyen CTH, Koito H, Shiojima I, Nishino I, Tsukaguchi H
Scientific reports 2018 Feb 20;8(1):3326

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Core Transcription Factors, MicroRNAs, and Small Molecules Drive Transdifferentiation of Human Fibroblasts Towards The Cardiac Cell Lineage.

Christoforou N, Chakraborty S, Kirkton RD, Adler AF, Addis RC, Leong KW
Scientific reports 2017 Jan 10;7:40285

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S100A6 Regulates Endothelial Cell Cycle Progression by Attenuating Antiproliferative Signal Transducers and Activators of Transcription 1 Signaling.

Lerchenmüller C, Heißenberg J, Damilano F, Bezzeridis VJ, Krämer I, Bochaton-Piallat ML, Hirschberg K, Busch M, Katus HA, Peppel K, Rosenzweig A, Busch H, Boerries M, Most P
Arteriosclerosis, thrombosis, and vascular biology 2016 Sep;36(9):1854-67

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Biliary tree stem cells, precursors to pancreatic committed progenitors: evidence for possible life-long pancreatic organogenesis.

Wang Y, Lanzoni G, Carpino G, Cui CB, Dominguez-Bendala J, Wauthier E, Cardinale V, Oikawa T, Pileggi A, Gerber D, Furth ME, Alvaro D, Gaudio E, Inverardi L, Reid LM
Stem cells (Dayton, Ohio) 2013 Sep;31(9):1966-79

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Characteristics of CD8+ T cell subsets in Chinese patients with chronic HIV infection during initial ART.

Jiao Y, Hua W, Zhang T, Zhang Y, Ji Y, Zhang H, Wu H
AIDS research and therapy 2011 Mar 25;8:15

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunocytochemistry of fixed and permeabilized HeLa cells stained with 20 µg/mL of Anti-Human Ki-67 eFluor® 660. Nuclei are stained with DAPI.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescence analysis of Ki-67 Monoclonal Antibody (20Raj1), eFluor™ 660, eBioscience™ was performed using 70% confluent log phase HeLa cells serum starved (SS) for 36 hours followed by serum release (SR) for 6 hours. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with Ki-67 Monoclonal Antibody (20Raj1), eFluor™ 660, eBioscience™ (Product # 50-5699-82) at 5 µg/mL concentration in 0.1% BSA, incubated at 4 degree celsius overnight (Panel a: Red). Nuclei (Panel b:Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Green) was stained with Alexa Fluor™ Plus 488 Phalloidin (Product # A12379, 1:500 dilution). Panel d represents the merged image showing speckle-like localization in the nucleus. Panel e represents serum starved cells (36 hours) having no Ki-67 expression. Panel f represents a mitotic cell among the HeLa control cells showing Ki-67 signal on the chromatin. The images were captured at 60X magnification.

Immunohistochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunohistochemical analysis of Ki-67 was performed using formalin-fixed paraffin-embedded human colon adenocarcinoma tissue sections. To expose the target protein, heat-induced epitope retrieval was performed on de-paraffinized sections using eBioscience™ IHC Antigen Retrieval Solution - High pH (10X) (Product # 00-4956-58) diluted to 1X solution in water in a decloaking chamber at 110 degree Celsius for 15 minutes. Following antigen retrieval, the sections were blocked with 2% normal goat serum in 1X PBS for 45 minutes at room temperature and then probed with or without Ki-67 Monoclonal Antibody (20Raj1), eFluor™ 660, eBioscience™ (Product # 50-5699-82) at 2 µg/mL concentration in 0.1% normal goat serum overnight at 4 degree Celsius in a humidified chamber. ReadyProbes™ Tissue Autofluorescence Quenching Kit (Product # R37630) was used to quench autofluorescence from the tissues. Nuclei were stained with DAPI (Product # D1306) and the sections were mounted using ProLong™ Glass Antifade Mountant (Product # P36984). The images were captured on EVOS™ M7000 Imaging System (Product # AMF7000) at 20X magnification and externally deconvoluted.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunohistochemical analysis of Ki-67 was performed using formalin-fixed paraffin-embedded human colon adenocarcinoma tissue sections. To expose the target protein, heat-induced epitope retrieval was performed on de-paraffinized sections using eBioscience™ IHC Antigen Retrieval Solution - High pH (10X) (Product # 00-4956-58) diluted to 1X solution in water in a decloaking chamber at 110 degree Celsius for 15 minutes. Following antigen retrieval, the sections were blocked with 2% normal goat serum in 1X PBS for 45 minutes at room temperature and then probed with or without Ki-67 Monoclonal Antibody (20Raj1), eFluor™ 660, eBioscience™ (Product # 50-5699-82) at 2 µg/mL concentration in 0.1% normal goat serum overnight at 4 degree Celsius in a humidified chamber. ReadyProbes™ Tissue Autofluorescence Quenching Kit (Product # R37630) was used to quench autofluorescence from the tissues. Nuclei were stained with DAPI (Product # D1306) and the sections were mounted using ProLong™ Glass Antifade Mountant (Product # P36984). The images were captured on EVOS™ M7000 Imaging System (Product # AMF7000) at 20X magnification. and externally deconvoluted.

Flow cytometry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Intracellular staining of CD3-stimulated PBMCs with Anti-Human CD19 PE (Product # 12-0199-80) and 0.125 µg of Anti-Human Ki-67 eFluor® 660 using the Foxp3/Transcription Factor Staining Buffer Set (Product # 00-5523-00). Total lymphocytes were used for the analysis.

Other assay

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

NULL

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

NULL

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

NULL

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

NULL

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

NULL

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Figure 2 Immunofluorescence characterization of transdifferentiated iCM (cardiac TF and microRNA). ( A ) Expression of ACTN2 and TNNT2 or ( B ) Expression of TNNT2 and Ki67 in HDF transdifferentiated for 2 weeks using induced expression of GATA4, TBX5, MEF2C, MYOCD, NKX2-5 and transfection with hsa-miR-1 and hsa-miR-133a. (Controls: M2rtTA only, microRNA only, cardiac TF only). ( C ) ACTN2. Panels on the right show varying degrees of cytoskeletal organization of ACTN2. ( D ) TNNT2. Panels on the right show varying degrees of cytoskeletal organization of TNNT2. ( E ) ACTN2 and GJA1. ( F ) TNNT2 and Ki67 expression in iCM. ( G ) VIM and MYH6/7. ( H ) TAGLN. ( I ) SMA. ( J ) MYH11. ( K ) ACTN2 expression in iCM 4 weeks following initiation of transdifferentiation. Panels on the right show varying degrees of cytoskeletal organization of ACTN2. ( L ) TNNT2 expression in iCM 4 weeks following initiation of transdifferentiation. Panels on the right show varying degrees of cytoskeletal organization of TNNT2.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Figure 6 Determining the effect of small molecule inhibitors on transdifferentiation efficiency. ( A ) Immunofluorescence of transdifferentiated cells (ACTN2/TNNT2) during exposure to small molecule inhibitors: Janus protein tyrosine kinase 1 (JAK1i), Sodium Butyrate (HDACi), SB431542 (TGFbetai), CHIR99021 (GSK3i). ( B ) Number of nuclei, ( C ) Number of ACTN2 + cells, and ( D ) Number of TNNT2 + cells (per mm 2 ). ( E ) Number of Ki67 + nuclei normalized to the total number of nuclei. ( F ) Number of ACTN2 + cells normalized to the total number of nuclei. ( G ) Number of TNNT2 + cells normalized to the total number of nuclei. Experiment performed in triplicate. 4 images were analyzed for each experiment. Error bar represents calculated standard deviation. Significant difference between two values was calculated using t-test (two-tailed distribution, two sample unequal variance).

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Figure 7 Determining the effect of protein ligands on transdifferentiation efficiency. ( A ) Immunofluorescence of transdifferentiated cells (ACTN2/TNNT2) during exposure to protein ligands: IGF1, EGF, NRG1, SDF1A. ( B ) Number of nuclei, ( C ) Number of ACTN2 + cells, and ( D ) Number of TNNT2 + cells (per mm 2 ). ( E ) Number of Ki67 + nuclei normalized to the total number of nuclei. ( F ) Number of ACTN2 + cells normalized to the total number of nuclei. ( G ) Number of TNNT2 + cells normalized to the total number of nuclei. Experiment performed in triplicate. 4 images were analyzed for each experiment. Error bar represents calculated standard deviation. Significant difference between two values was calculated using t-test (two-tailed distribution, two sample unequal variance).

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Figure 12 PR PEG10 influences the proliferation of HEK293T cells. In the cell proliferation assay, HEK293T ( a ) and HaCaT cells ( b ) were stained with FITC-conjugated anti-Ki67 antibody, which was detected using a flow cytometer (FACS). Significance values were determined as compared to mock control, otherwise they are indicated. Note: * p 0.05). Error bars represent SD ( n >= 2).

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Figure 1 PfRH5 vaccination with the ChAd63-MVA platform induces greater activation of total circulating CD8 + and CD4 + T cell populations, while the protein/AS01 B platform elicits a more sustained increase in cTfh-phenotype cells PBMCs from days 0, 7, 14, and 63 were stained ex vivo and analyzed using the gating strategies shown in Figures S1-S3 . (A and B) Increases at day 63 in activated CD38 + Ki67 + cells within total CD8 + and CD4 + T cells (A) or Th1 (CXCR3 + CCR6 - ), Th2 (CXCR3 - CCR6 - ), and Th17 (CXCR3 - CCR6 + ) CD4 + T cell subsets (B) were compared between platforms following subtraction of day 0 CD38 + Ki67 + frequencies in paired samples. (C-E) Frequencies of total cTfh cells defined as CXCR5 + (C) or CXCR5 + PD1 + (D) cells within the CD45RA - CD4 + T cell population were compared between platforms and the frequency of cTfr cells within total cTfh cells (defined as the CD25 + Foxp3 + subset) (E). All of the available samples are plotted (ChAd63-MVA/protein/AS01 B ): day 0, n = 15/54; day 7, n = 15/24; day 14, n = 15/54; day 63, n = 12/20. For intra-trial comparisons (E), only vaccinees with all 4 time points were analyzed: ChAd63-MVA, n = 12, protein/AS01 B , n = 17. Comparisons were performed with Mann-Whitney tests (between trials) or Friedman tests with Dunn's correction for multiple comparisons (within trials comparing day 0 to post-vaccination time points). *p < 0.5, **p < 0.01, and ***p < 0.001. In all of the panels, each point represents a vaccinee.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Figure 2 High Autophagy Level of Intrahepatic T Cells Is Not a Result of a Difference in Differentiation Status or Recent Proliferation (A) Example plot of CD45RA versus CCR7 staining (CD8 + T cells) from a PBMC or IHL sample and summary data for LC3 staining of CD8 + T cell memory subsets (PBMC, 9; and IHL, 15, biological replicates; box whisker, Tukey). (B) Comparison of LC3 staining of CD8 + T cell memory subsets between paired PBMC and IHL samples (9 biological replicates; box whisker, Tukey; outliers shown as dots). (C) Ex vivo CD8 + T cell Ki67 expression. (D and E) Example plots (CD8 + T cells, PBMC) (D) and summary data for LC3 staining on Ki67 - and Ki67 + CD8 + T cells (E) in PBMCs and IHLs ex vivo (10 biological replicates) or after anti-CD3/CD28 stimulation (overnight, 0.5 mug/mL each; three biological replicates) in PBMCs. (F) Histograms showing the dilution of CellTrace Violet (CTV), LC3 staining, and co-staining of LC3 and CTV on CD8 + T cells after 5 days of stimulation with anti-CD3/CD28 (red), compared with that without stimulation (gray) or without CTV staining (black; two representative biological replicates of five, PBMCs). Cells were treated with bafA1 (A-F). Friedman test (ANOVA) with Dunn's post hoc test for pairwise multiple comparisons (A and B). Mann-Whitney t test (C and E). * p < 0.05, ** p < 0.005, *** p < 0.001.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Figure 4 Streptomyces Levis ABRIINW111 can alter the apoptosis and proliferation of colon cancer cells. SW480 was treated with indicated concentrations of metabolites for 48 h and was evaluated by flow cytometry (FACS) for Caspase3 and Ki-67 expression. (a) FACS profile of Caspase 3 expression: Control cells (a) 100 ng ml -1 (b) 500 ng ml -1 (c) 1000 ng ml -1 (d).(b) Mean (SD) proportion of Caspase 3 expression with an indicated concentration of metabolites. (c) FACS profile of Ki67 expression: Control cells (a) 100 ng ml -1 (b) 500 ng ml -1 (c) 1000 ng ml -1 (d). (d) Mean (SD) proportion of Ki67 expression with the indicated concentrations of metabolites. Values are demonstrated as mean +- SD from 4 independent experiments (P_Value