Cell activation and viability were assessed following treatment with either negative control GapmeR (Control A) or positive control GapmeR (MALAT1) at 300 nM and 500 nM concentrations. Cells were washed with phosphate buffered saline + 0.1% bovine serum albumin and stained with Aqua fluorescence reactive dye (Thermo Fisher Scientific, Inc. Waltham, MA, USA) to assess viability, and with the following antibodies to assess activation: CD69-FITC clone L78, CD38-PE-Cy7 clone HB7, and HLA-DR-APC-Cy7 clone L243 (BD Biosciences, Inc., San Jose, CA, USA). Custom-made CD4-APC clone SK3+SK4 (BD Biosciences, Inc., San Jose, CA, USA) was used to stain CD4+ T cells to exclude small percentage of contaminants from the analysis. The data was acquired using FACS Canto II instrument (BD Biosciences, Inc., San Jose, CA, USA) and analyzed using FlowJo v10 software (FlowJo, LLC, Ashland, OR, USA). Gating strategy is shown in Figure S1A .
Aqua fluorescence reactive dye
Manufactured by Thermo Fisher Scientific
Sourced in United States
Aqua fluorescence reactive dye is a laboratory product designed to detect and quantify proteins in various applications. It is a water-soluble dye that binds to proteins, emitting a fluorescent signal that can be measured using a fluorescence spectrometer or plate reader. The dye provides a simple and sensitive method for protein quantification and is commonly used in protein assays, Western blotting, and other protein analysis techniques.
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Lab products found in correlation
Facs canto 2 instrument, by BD (1 mentions)
Cd38 pe cy7 clone hb7, by BD (1 mentions)
Celltrace calcein violet, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Vitoss, by Stryker (1 mentions)
Collagenase, by STEMCELL (1 mentions)
2 protocols using aqua fluorescence reactive dye
1
Characterizing T Cell Activation and Viability
2
Enrichment of Mesenchymal Stem Cells from Bone Marrow
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Unprocessed samples of IC-BM and VB-BM aspirates were seeded onto Vitoss™ (Stryker® UK Limited, Berkshire, UK) as described before [29 (link)]. Briefly, 400μl of BM sample was added into 100mm3 of Vitoss then incubated at 37°C with gentle rocking for 3 hours to enhance the cell attachment. Each BM sample was used to seed Vitoss in duplicate to examine the MSC attachment and survival using microscopy and flowcytometry respectively. The scaffolds were next rinsed with PBS to remove red blood cells then moved into culture plates in the StemMACS MSC Expansion media and cultured for 2 weeks. After culture, for microscopy, the scaffolds were examined for cell attachment using a SP2 TCS confocal laser scanning microscope (Leica, Buckinghamshire, UK). The DAPI (Thermo Fisher Scientific) and Phalloidin (Sigma-Aldrich) dyes were used to detect cell nuclei and actin, respectively. For flowcytometry-dependent characterisation, the scaffolds were digested using 0.25% collagenase (Stem Cell Technologies). and the released cells were characterised for the surface phenotype of MSCs as previously described [29 (link)]. The released cells were stained using CD45, CD90 and CD73 antibodies (BD Biosciences) as well as live/dead markers, CellTrace calcein violet and Aqua-fluorescence reactive dye (Thermo Fisher Scientific), to identify live MSCs.
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