Cells were gently dissociated using the Multi Tissue Dissociation Kit (Miltenyi Biotec) and incubated using the LIVE/DEAD Fixable Green Dead cell stain kit (Thermo Fisher Scientific). Cells were fixed in inside fix solution (Miltenyi Biotec) and stained with primary antibodies diluted in inside perm solution (Miltenyi Biotec). Conjugated primary antibodies used were α-actinin-VioBlue (1:10; 130-106-996; Miltenyi Biotec) and KI67-APC (1:10; 130-111-761; Miltenyi Biotec). As a control, universal isotype control antibodies (REA; Miltenyi Biotec) were used. Media and washes were collected to obtain a complete representation of cell loss. The samples were analyzed using the FACS Canto system (BD Bioscience) and FlowJo software.
Inside fix solution
Manufactured by Miltenyi Biotec
Sourced in Italy
The Inside fix solution is a reagent used in cell analysis and cytometry applications. It is designed to permeabilize cell membranes, allowing for the intracellular staining and detection of specific cellular components or proteins. The solution facilitates the access of antibodies or other detection probes to the interior of cells, enabling the analysis of intracellular targets.
Automatically generated - may contain errors
Lab products found in correlation
Inside perm solution, by Miltenyi Biotec (3 mentions)
Live dead fixable green dead cell stain kit, by Thermo Fisher Scientific (2 mentions)
Facscanto system, by BD (2 mentions)
Multi tissue dissociation kit, by Miltenyi Biotec (2 mentions)
Ki67 apc, by Miltenyi Biotec (1 mentions)
Vioblue, by Miltenyi Biotec (1 mentions)
Anti rabbit af488, by Thermo Fisher Scientific (1 mentions)
Rabbit anti sirt1 h300, by Santa Cruz Biotechnology (1 mentions)
4 6 diamidino 2 phenylindole dapi containing mounting solution, by Merck Group (1 mentions)
Apotome 2 acquisition system, by Zeiss (1 mentions)
Anti mouse af488, by Thermo Fisher Scientific (1 mentions)
3 protocols using inside fix solution
1
Multiparametric Flow Cytometry Analysis
2
Viability and Phenotypic Analysis of Cells
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Cells were gently dissociated (multi-tissue dissociation kit, Miltenyi Biotec, 130-110-204) and incubated with LIVE/DEAD fixable green dead cell stain kit (Thermo Fisher Scientific, L23101). This kit contains a fixable fluorescent dye that binds to amines. In viable cells, this amine-reactive dye binds amines on the outer cell surface, as opposed to dead cells in which the dye can additionally bind internal amines due to membrane disruption. After staining, cells were fixed (inside fix solution, Miltenyi Biotec, 130-090-477) and stained with primary antibodies (diluted in inside perm solution, Miltenyi Biotec, 130-090-477). ACTN1-VioBlue (Miltenyi Biotec, 130-127-354, 1:10) and cardiac troponin T VioBlue (Miltenyi Biotec, 130-120-402, 1:10) were used as conjugated antibodies. As a control, universal isotype control antibodies (REA, Miltenyi Biotec, 130-096-932) were used. Media and washes were collected to obtain a complete representation, including detached cells. The samples were analyzed using a FACS Canto system (BD Biosciences, FACSDiva software 6.0) and FlowJo software (BD Biosciences, v10).
3
Immunofluorescent Analysis of SIRT1 and NF-κB
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Cells were fixed using InsideFix Solution (Miltenyi Biotec, Bologna, Italy) and incubated overnight at 4 °C with primary antibodies diluted in InsidePerm solution (Miltenyi, Bologna, Italy). The antibodies used were rabbit anti-SIRT1(H300) (1:400; Santa Cruz Biotechnologies, Santa Cruz, CA, USA, Cat. No. sc-15404) and rabbit anti-acetyl-NF-kB p65 (Lys310) (1:30; Cell Signaling, Danvers, MA, USA, Cat. No. 3045). After washing, cells were incubated with secondary antibodies, diluted in InsidePerm solution, for 45 min RT. The secondary antibodies used were AF488-anti-mouse (1:300; Thermofisher Scientific, Waltham, MA, USA) and AF488-anti-rabbit (1:300; Thermofisher Scientific, Waltham, MA, USA). After washing, slides were mounted with 4′,6-diamidino-2-phenylindole (DAPI)-containing mounting solution (Sigma-Merck, Darmstadt, Germany). Digital images were captured with a Zeiss Observer.Z1 microscope equipped with the Apotome.2 acquisition system (Zeiss, Oberkochen, Germany). The number of immunopositive cells with nuclear SIRT1 was determined by cell counting in at least five randomly selected fields/well.
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