Cd44 vioblue

Manufactured by Miltenyi Biotec

Sourced in Germany

CD44-VioBlue is a monoclonal antibody conjugated with the fluorescent dye VioBlue. It is designed for the detection and analysis of CD44 expression in flow cytometry applications.

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4 protocols using cd44 vioblue

Ovalbumin-Loaded Silica Nanoparticles for Immunotherapy

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Rhodamine B isothiocyanate
(RITC), (3-aminopropyl)trimethoxysilane (APTMS), cethyltrimethylammonium
bromide (CTAB), ammonium hydroxide, tetraethylorthosilicate (TEOS),
and ovalbumin (OVA) were purchased from Sigma-Aldrich (St. Louis,
MO, USA). HCl, methanol, and ethyl acetate were purchased from SamChun
Chemical (Seoul, Korea). Bovine serum albumin (BSA) was purchased
from Millipore (Billerica, MA, USA), CpG oligodeoxynucleotide was
purchased from Bioneer (Daejeon, Korea). Recombinant murine GM-CSF
was purchased from Peprotech (Rocky Hill, NJ, USA). Antibodies against
the following proteins were used: CD11c-APC, MHC class-II-FITC, CD86-Vioblue,
MHC class-I(H-2K^b)/SIINFEKL-PE-Vio770, CD3e-FITC, CD4-PE-Vio770,
CD8-APC, IFN-γ-FITC, CD44-Vioblue, and CD62L-PE were purchased
from Miltenyi Biotec (Bergisch Gladbach, Germany) and H-2K^b/SIINFEKL tetramer-PE was purchased from MBL life science (Woburn,
MA). IL-12 and TNF-α ELISA kits were purchased from BD science.

Cha B.G., Jeong J.H, & Kim J. (2018). Extra-Large Pore Mesoporous Silica Nanoparticles Enabling Co-Delivery of High Amounts of Protein Antigen and Toll-like Receptor 9 Agonist for Enhanced Cancer Vaccine Efficacy. ACS Central Science, 4(4), 484-492.

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CD24/CD44 Expression Analysis Protocol

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One million cells were stained in 100 µL of Zombie Aqua (Fixable Viability Kit BioLegend; San Diego, CA, USA) for 20 min at room temperature. Zombie Aqua was removed and 20 µL of antibody dilution was added to each sample, which was then incubated on ice for 10–15 min.
Antibody pairs:

CD24 APC (REA832, MiltenyiBiotec, 1:20 dilution), CD44 Vioblue (REA690, Miltenyi Biotec, 1:5 dilution) used in Fig. 1f

FITC Mouse Anti-Human CD24 (BD Biosciences; Franklin Lakes, NJ, USA, 1:5 dilution), PE Mouse Anti-Human CD44 (BD Biosciences, 1:5 dilution) used in Fig. 3d and Supplementary Figs. 1b–d and 3f,g

Cells were washed with 200 µL FACs buffer (PBS with 1% FBS), pelleted and then resuspended in 100 µL 2% PFA in FACs buffer. For acquisition, cells were re-suspended in 300 µL FACS buffer for flow acquisition. Doublet discrimination and live cell gates were used to identify the cells of interest, and quadrant gates were set according to the fluorescence minus one controls (FMO) (Supplementary Fig. 7).

Jewer M., Lee L., Leibovitch M., Zhang G., Liu J., Findlay S.D., Vincent K.M., Tandoc K., Dieters-Castator D., Quail D.F., Dutta I., Coatham M., Xu Z., Puri A., Guan B.J., Hatzoglou M., Brumwell A., Uniacke J., Patsis C., Koromilas A., Schueler J., Siegers G.M., Topisirovic I, & Postovit L.M. (2020). Translational control of breast cancer plasticity. Nature Communications, 11, 2498.

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Immunophenotypic analysis of stem cells

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Immunophenotypic analyses with flow cytometry were performed according to manufacturer’s recommendations. Briefly, 1 × 10⁶ cells per sample (Passage 3–4) were centrifuged at 300× g and resuspended in 100 µl of buffer. Afterwards, 10 µl of the respective antibodies, CD44-VioBlue, CD73-APC, CD90-PE, CD105-FITC, CD146-PE-Vio770 and CD271-APC-Vio770 (Miltenyi Biotec, Bergisch Gladbach, Germany) and a cocktail of CD14/CD20/CD34/CD45-PerCP (Miltenyi Biotec, Bergisch Gladbach, Germany), was added to cell suspension and incubated for 10 min in the dark in the refrigerator. Then, cells were washed with 2 mL of buffer and centrifuged. Supernatant was aspirated, and the final sediment was resuspended in buffer for flow cytometry analysis. Similarly, respective iso-types controls were used to assess background fluorescence and non-specific binding of anti-bodies to cells. All data were acquired using a MACSQuant Analyzer 10 (Miltenyi Biotec, Bergisch Gladbach, Germany) and further analyzed by MACS Quantify software (Miltenyi Biotec, Bergisch Gladbach, Germany).

Culenova M., Nicodemou A., Novakova Z.V., Debreova M., Smolinská V., Bernatova S., Ivanisova D., Novotna O., Vasicek J., Varga I., Ziaran S, & Danisovic L. (2021). Isolation, Culture and Comprehensive Characterization of Biological Properties of Human Urine-Derived Stem Cells. International Journal of Molecular Sciences, 22(22), 12503.

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Characterizing MSC Phenotype in Normoxic and Hypoxic Conditions

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To confirm MSCs phenotype of cells grown in normoxic and hypoxic conditions were analyzed by flow cytometry. Antibodies against the following human antigens were used: CD105-FITC (Miltenyi Biotect, Bergisch Gladbach, Germany, cat# 130-112-327, 1:50), CD90-FITC (Miltenyi Biotec, cat# 130-114-901, 1:50), CD44-VioBlue (Miltenyi Biotec, cat# 130-113-906, 1:50), CD73-APC (Miltenyi Biotec, cat# 130-111-909, 1:50), MSC Phenotyping Cocktail-PE (CD34, CD14, CD19, CD45, Miltenyi Biotec cat# 130-125-285, dilution according to the manufacturer’s instructions). Briefly, cells were seeded at a concentration of 10⁴ cells/cm² and maintained in culture until they reached 80%–90% confluence. Then cells were trypsinized, surface labeled, washed and then analyzed using a flow cytometer (Miltenyi Biotech). The data were analyzed with MACSQuantify™ Software.

de Laorden E.H., Simón D., Milla S., Portela-Lomba M., Mellén M., Sierra J., de la Villa P., Moreno-Flores M.T, & Iglesias M. (2023). Human placenta-derived mesenchymal stem cells stimulate neuronal regeneration by promoting axon growth and restoring neuronal activity. Frontiers in Cell and Developmental Biology, 11, 1328261.

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