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Aldefluor staining kit

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The ALDEFLUOR staining kit is a laboratory tool used to identify and isolate cells with high aldehyde dehydrogenase (ALDH) activity. ALDH is an enzyme associated with stem and progenitor cells. The kit provides reagents and protocols for the detection and analysis of ALDH-positive cells using flow cytometry or fluorescence-activated cell sorting (FACS).

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Lab products found in correlation

Facscalibur, by BD (3 mentions) Facsverse flow cytometer, by BD (2 mentions) Fixation buffer, by BioLegend (1 mentions) Monensin, by Merck Group (1 mentions) Cellquest pro software, by BD (1 mentions) Facsaria, by BD (1 mentions) Facscanto 2 cytometer, by BD (1 mentions) Facsdiva software, by BD (1 mentions) Apc conjugated anti cd11c, by BD (1 mentions) Anti mouse cd11c microbeads, by Miltenyi Biotec (1 mentions) Aldh inhibitor deab, by STEMCELL (1 mentions) Aldered aldh detection assay, by Merck Group (1 mentions) Sytox blue dead cell stain, by Thermo Fisher Scientific (1 mentions) Interleukin 4 (il 4), by Thermo Fisher Scientific (1 mentions) Gm csf, by Thermo Fisher Scientific (1 mentions) Liberase dl, by Roche (1 mentions) Dnase 1, by Roche (1 mentions) Cd11c microbeads, by Miltenyi Biotec (1 mentions) Fixation permeabilization solution, by Thermo Fisher Scientific (1 mentions) Ionomycin, by Merck Group (1 mentions)

21 protocols using aldefluor staining kit

1

Measuring ALDH Activity and Cytokine Production in Cells

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ALDH activity in individual cells was estimated using ALDEFLUOR staining kits (Stemcell Technologies) as previously described14 (link). Cells were stained with indicated mAbs conjugated to various fluorochromes in the presence of anti-CD16/CD32 mAb. Intracellular staining of Foxp3 was performed using mouse/rat Foxp3 staining sets (eBioscience). For intracellular cytokine staining, CD4+ T cells obtained from cultures were restimulated for 5 h with PMA (50 ng/ml) and ionomycin (750 ng/ml) (both from Calbiochem). Monensin (3 μM; Sigma-Aldrich) was added to the cultures for the last 2 h. After surface staining, the cells were fixed with Fixation Buffer (BioLegend), and intracellular cytokine staining was performed according to the manufacturer’s protocol. Analysis was performed on a FACSAria or FACSCalibur with CellQuest Pro software (BD Biosciences).
Yokota-Nakatsuma A., Ohoka Y., Takeuchi H., Song S.Y, & Iwata M. (2016). Beta 1-integrin ligation and TLR ligation enhance GM-CSF–induced ALDH1A2 expression in dendritic cells, but differentially regulate their anti-inflammatory properties. Scientific Reports, 6, 37914.
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2

Quantifying RALDH Activity in DCs

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RALDH activity in DCs was measured using ALDEFLUOR staining kits (StemCell Technologies), according to the manufacturer’s protocol. Briefly, 106 DCs/mL were resuspended in ALDEFLUOR assay buffer containing Aldefluor reagent (1.5 µM final concentration) in the presence or absence of DEAB (diethylaminobenzaldehyde, 15 µM) and then DCs were incubated 45 min at 37 °C. After Aldefluor staining, the cells were incubated for 30 min at 4 °C with APC-conjugated anti-CD11c. ALDEFLUOR-reacted cells were analysed by flow cytometry using a FACS Canto II cytometer (BD), and data were analysed using FACS Diva software (BD).
Doñas C., Neira J., Osorio-Barrios F., Carrasco M., Fernández D., Prado C., Loyola A., Pacheco R, & Rosemblatt M. (2021). The demethylase inhibitor GSK-J4 limits inflammatory colitis by promoting de novo synthesis of retinoic acid in dendritic cells. Scientific Reports, 11, 1342.
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3

Measurement of RALDH Activity in BMDCs

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Immature BMDCs were pre-incubated with epinephrine, propranolol followed by epinephrine or vehicle control and activated with LPS for 24 hrs. After maturation cells were harvested and RALDH activity in individual cells was measured using Aldefluor staining kit (StemCell Technologies, Grenoble, Fr), according to the manufacture's protocol with modifications. Briefly, cells were suspended at 106 cells/ml in Aldefluor assay buffer containing activated Aldefluor reagent (1.5 µM) with or without the RALDH inhibitor diethylaminobenzaldehyde (DEAB, 15 µM) were incubated for 40 min at 37°C. The cells were subsequently stained with APC conjugated anti-CD11c (BD Biosciences) in ice-cold Aldefluor assay buffer. Aldefluor reactive cells were detected using a FACSCalibur flow cytometer. Cell viability was determined by flow cytometry with 7AAD exclusion.
Nijhuis L.E., Olivier B.J., Dhawan S., Hilbers F.W., Boon L., Wolkers M.C., Samsom J.N, & de Jonge W.J. (2014). Adrenergic β2 Receptor Activation Stimulates Anti-Inflammatory Properties of Dendritic Cells In Vitro. PLoS ONE, 9(1), e85086.
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4

Dendritic Cell Activation Protocol

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Spleens were pretreated with collagen (Sigma-Aldrich) for 30 min at 37°C. Single cell suspensions were incubated with anti–mouse CD11c microbeads (Miltenyi Biotec) and positively selected on MACS columns. Cells were further purified by cell sorting by gating on CD11c+MHC IIhi. DCs were cultured with 500 pg/ml GM-CSF for 48 h. These cells were resorted for identical expression of 4-1BB, and 2–3 × 105 DCs were further cultured with 2.5 µg/ml zymosan in the presence of 5 µg/ml control rat IgG (KLH/G1-2-2) or 5 µg/ml anti–4-1BB (3H3) for another 24 h. The activity of RALDH was determined by the ALDEFLUOR staining kit (STEMCELL Technologies).
Madireddi S., Eun S.Y., Lee S.W., Nemčovičová I., Mehta A.K., Zajonc D.M., Nishi N., Niki T., Hirashima M, & Croft M. (2014). Galectin-9 controls the therapeutic activity of 4-1BB–targeting antibodies. The Journal of Experimental Medicine, 211(7), 1433-1448.
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5

Aldehyde Dehydrogenase Activity Profiling

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The presence of cells with aldehyde dehydrogenase activity (ALDHbright) was assessed using the Aldefluor staining kit (StemCell Technologies, Cambridge, MA) following the manufacturer’s protocol. Single cell suspensions from either conjunctiva or CLN were incubated in the dark for 1h at 37°C in Aldefluor assay buffer containing activated Aldefluor substrate, with or without the ALDH inhibitor, diethylaminobenzaldheyde (DEAB) in ice-cold Aldefluor assay buffer, resuspended in Aldefluor assay buffer containing propidium iodide, and kept on ice until immediate analysis on a FACS Canto II System. Data were analyzed using FlowJo software version 10 (TreeStar Inc., Ashland, OR). For each sample, the total percentage of positive cells was calculated after subtraction of test tube results from control results (cells stained with DEAB). After live/dead staining and singlet discrimination, aldefluor positive cells were gated and analyzed.
Bian F., Xiao Y., Barbosa F.L., de Souza R.G., Hernandez H., Yu Z., Pflugfelder S.C, & de Paiva C.S. (2019). Age-associated Antigen-Presenting Cell Alterations Promote Dry-Eye Inducing Th1 cells. Mucosal immunology, 12(4), 897-908.
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6

Aldehyde Dehydrogenase Activity Profiling

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The presence of cells with aldehyde dehydrogenase activity (ALDHbright) was assessed using the Aldefluor staining kit (StemCell Technologies, Cambridge, MA) following the manufacturer’s protocol. Single cell suspensions from either conjunctiva or CLN were incubated in the dark for 1h at 37°C in Aldefluor assay buffer containing activated Aldefluor substrate, with or without the ALDH inhibitor, diethylaminobenzaldheyde (DEAB) in ice-cold Aldefluor assay buffer, resuspended in Aldefluor assay buffer containing propidium iodide, and kept on ice until immediate analysis on a FACS Canto II System. Data were analyzed using FlowJo software version 10 (TreeStar Inc., Ashland, OR). For each sample, the total percentage of positive cells was calculated after subtraction of test tube results from control results (cells stained with DEAB). After live/dead staining and singlet discrimination, aldefluor positive cells were gated and analyzed.
Bian F., Xiao Y., Barbosa F.L., de Souza R.G., Hernandez H., Yu Z., Pflugfelder S.C, & de Paiva C.S. (2019). Age-associated Antigen-Presenting Cell Alterations Promote Dry-Eye Inducing Th1 cells. Mucosal immunology, 12(4), 897-908.
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7

Quantifying Aldehyde Dehydrogenase Activity in Dendritic Cells

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Cell aldehyde dehydrogenase (ALDH) activity was determined by using the ALDEFLUOR staining kit (Stemcell Technologies, Cambridge, UK) according to the manufacturer's instructions. In brief, DCs treated with or without 0.1 μM citral were resuspended at 106 cells/ml in ALDEFLUOR assay buffer containing activated ALDEFLUOR substrate and incubated at 37°C for 30 min. ALDH inhibitor diethylaminobenzaldehyde (DEAB) was used as negative control. ALDEFLUOR-reactive cells were detected by flow cytometry in the FITC channel.
Fu H., Jangani M., Parmar A., Wang G., Coe D., Spear S., Sandrock I., Capasso M., Coles M., Cornish G., Helmby H, & Marelli-Berg F.M. (2019). A Subset of CCL25-Induced Gut-Homing T Cells Affects Intestinal Immunity to Infection and Cancer. Frontiers in Immunology, 10, 271.
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8

Measuring RALDH Enzyme Activity

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RALDH enzyme activity was measured using ALDEFLUOR staining kit (StemCell Technologies, Vancouver, Canada), according to the manufacturer’s protocol with some modifications. Briefly, cells were suspended in ALDEFLUOR assay buffer containing the activated ALDEFLUOR substrate and incubated for 45 min at 37 °C. They were then stained with cell surface antibodies and ALDEFLUOR+ cells were measured by flow cytometry.
Zheng J., Taylor B., Dodge J., Stephans A., Zheng S.G., Chen Q, & Chen X. (2019). RADIATION AND HOST RETINOIC ACID SIGNALING PROMOTE THE INDUCTION OF GUT-HOMING DONOR T CELLS AFTER ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 20(1), 64-74.
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9

ALDEFLUOR Assay for RALDH1 Activity

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Cell RALDH1 activity was determined by using the ALDEFLUOR staining kit (STEMCELL Technologies Inc.) (5 (link)). Briefly, cells were resuspended at 106 cells/ml in ALDEFLUOR assay buffer, containing activated ALDEFLUOR substrate with or without the inhibitor DEAB, and incubated at 37°C for 30 min. ALDEFLUOR-reactive cells were detected in the FITC channel.
Jie Z., Liang Y., Yi P., Tang H., Soong L., Cong Y., Zhang K, & Sun J. (2017). Retinoic acid regulates immune responses by promoting IL-22 and modulating S100 proteins in viral hepatitis. Journal of immunology (Baltimore, Md. : 1950), 198(9), 3448-3460.
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10

Measuring ALDH Activity in LP Cells

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The aldehyde dehydrogenase (ALDH) activity of the LP cells was detected using an ALDEFLUOR staining kit (StemCell Technologies, Canada). Briefly, the LP cells in ALDEFLUOR buffer containing ALDH substrate were incubated at 37°C for 45 min in the dark. After washing with the buffer and centrifugation, the cell pellet was suspended in ice-cold ALDEFLUOR buffer, add and incubate with biotin-conjugated monoclonal anti-CD11c, anti-CD11b and anti-Siglec-F, and then detected by flow cytometry[15 (link)]. The ALDH activity was measured by the difference values of mean fluorescence intensity (MFI) of the LP cells before or after treatment with 300 μM ALDH inhibitor DEAB (StemCell Technologies).
Chen H.H., Sun A.H., Ojcius D.M., Hu W.L., Ge Y.M., Lin X., Li L.J., Pan J.P, & Yan J. (2015). Eosinophils from Murine Lamina Propria Induce Differentiation of Naïve T Cells into Regulatory T Cells via TGF-β1 and Retinoic Acid. PLoS ONE, 10(11), e0142881.
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