Aldefluor kit pdf
Dysregulation in the secretion of these factors can lead to inflammation and development of colitis. Aldefluor assay The Aldefluor Kit (Stem Cell Technologies, Durham, NC, USA) was used to detect stem cells with high ALDH1 enzyme activity according to the manufacturer’s instructions. The second aim of this thesis was to test this kit for new cancer stem cell lines with a multilabel reader and to visually capture the fluorometric signal. This enzyme converts a nonfluorescent sub-strate (an aminoacetaldehyde) into a fluorescent product (an aminoacetate) that is retained within living cells with an intact membrane. ALDH activity was measured using the Aldefluor kit (Stem Cell Technologies Inc., Vancouver, Canada) as described previously (6).
Different mechanisms contribute to intratumor heterogeneity, including genetic mutations, the microenvironment, and the existence of subpopulations of cancer cells with increased renewal capacity and the ability to recapitulate the heterogeneity found in primary tumors, which are referred to as cancer stem cells (CSCs). 1–4 This difference was also seen in our own cell cultures especially when the colon cancer cell lines were grown in low or high densities. The buffer is necessary to maintain ALDEFLUOR™ reaction product inside the cell, allowing detection of the ALDH-bright population.
ALDEFLUOR (Stem Cell Technologies), with or without the ALDH inhibitor diethylaminobenzaldehyde (DEAB; control) according to the manufacturer’s instructions (14). ALDEFLUOR assay kit was purchased from Stem Cell Technology (#01700) and was performed following the manufacturer’s protocol with only a few modifications. Aldefluor Kit (StemCell Technologies) according to the manufacturer’s instruction. The cells were incubated with Aldefluor substrate at 37 ℃ for 30 minutes and pelleted at 250xg for 5 mins. To examine ALDH1 expressing cells, ALDEFLUOR kit (Stem Cell Technologies) was used to isolate the population with high ALDH1 enzymatic activity as described before . ALDH1 activity was evaluated using the Aldefluor kit (Stem Cell Technologies, Vancouver). Pre-warm cell culture medium, Trypsin-EDTA (Catalog #07901), D-PBS (Catalog #37350) and all reagents in the ALDEFLUOR® Kit (Catalog #01700) to room temperature (15°C – 25°C). Cells were trypsinized to remove cells from the cell culture flask two days after seeding, washed and resuspended in 1 mL Aldefluor assay buffer.
assessed using the Cell-Titre Blue Kit after 48 h treatment.
The cancer stem cell (CSC) theory posits that a small population of cells with stem cell-like features is responsible for tumor growth, resistance, and recurrence in many malignancies. activity was tested by using ALDEFLOUR assay kit (STEMCELL Technologies) according to manufacturer’s manual. For these control tubes, place 1ml cell suspension into #1 and #4, but 2 ml into tube #2. Briefly, 1×106 NB cells or PDX-dissociated cells were resuspended in 1 ml ALDEFLUOR assay buffer. Dead cells were stained with 7-AAD Viability Staining Solution (BioLegend) or Fixable Viability Stain (FVS) 780 (BD Biosciences). is a global biotechnology company that develops, manufactures and sells products and provides services that support academic and industrial scientists. It is excluded by viable cells but can penetrate cell membranes of dying or dead cells.
Propidium iodide (PI) intercalates into double-stranded nucleic acids and fluoresces. ALDEFLUOR assay The ALDEFLUOR kit (Stem Cell Technologies, Vancouver, Canada) was used to identify and isolate cancer stem cells from the human lung cancer cell line H460 and freshly harvested primary tumor cells according to the manufacturer’s instructions. Furthermore, the frequency of ALDHbright cells was determined by this assay in SDCs after treatment with cisplatin or/and ATRA.
The amount of fluorescent ALDH reaction product that accumulates in the cells directly correlates to the ALDH activity in these cells. virtue of increased aldehyde dehydrogenase 1 (ALDH1) activity as assessed by the ALDEFLUOR assay and cancer stem cell-like features such as chemo-resistance, irradiation-resistance and tumor-initiating were confirmed in cell culture and in animal models. The ALDEFLUOR staining system allows for the identification and isolation of stem/progenitor cells based on their ALDH enzymatic activity, rather than their cell surface phenotype. Briefly, the cells were incubated in the ALDEFLUOR assay buffer containing ALDH substrate (BAAA, 1 mmol/l per 1 × 10 6 cells), and incubated for 40 min at 37 °C. The ALDEFLUOR Kit (STEMCELL Technologies, 01700) was used to measure the ALDH enzymatic activity in callus cells following the manufacturer’s protocol. Our study identified an aldefluor positive stem cell (CASC) population in adult human hearts. Therefore, the percentage of ALDHhigh cells in the control gate correspond always to 0.01% of total. CD45 is a leukocyte specific tyrosine phosphatase important for T cell development and antigen receptor signaling.
Then, 1 × 106 live cells were counted using a Trypan blue assay.
Two sets of tubes were labeled as sample and control for each cell line to be tested. While there is sufficient ALDEFLUOR™-buffer contained within the ALDEFLUOR™ kit, additional buffer may be required for certain immunolabeling procedures. Here, we present a method called ALDEFLUOR assay used for the identification, evaluation, and isolation of normal, cancer stem and progenitor cells. Aldefluor® The protocol was performed according to Aldefluor® guidelines and adapted to the cell type employed in this study.
ALDEFLUOR st aining of pancreatic cancer cells demonstrated that 27% of th e cells were ALDH positive. Thus PI staining is included in immunofluorescent staining protocols to identify dead cells.
This theory could be a useful paradigm for designing innovative targeted drug therapies. 4.2 Assemble all necessary supplies and allow kit reagents to come to room temperature (RT), 18˚ to 22˚C before use. on the immune detection reagents of independent research and development, the main products include ELISA kit, CLIA kit, protein, antibody, mark kit and other related reagents, which a total of more than 20000 products!
Briefly, trypsinized cells were pelleted and resuspended in Aldefluor reagent at a density of 1 × 106 /ml. RALDH-expressing cells were stained with the ALDEFLUOR Kit (Stem Cell Technologies, Vancouver, Canada), according to the manufacturer’s instructions. ALDEFLUOR Assay The fluorogenic dye-based ALDEFLUOR assay kit (StemCell Technologies) was used to detect the activity of ALDH as previously described[4, 12]. Purified HNSCC patient tumor cells were stained with propidium iodide and sorted by FACS to eliminate dead cells. cells by flow cytometry using the ALDEFLUOR reagent to measure ALDH enzymatic activity [6, 7]. tor™ Bisulfite Modification kit (Active Motif North America) according to the manufacturer’s instruction. Briefly, cells were incubated in ALDEFLUOR assay buffer containing ALDH substrate (1 Amol/L per 1 106 cells). Staining for ALDH activity was performed as previously reported using Aldefluor reagent kit (#01700; STEMCELL Technologies).
the kit, was added to the “control” tube.
ALDEFLUOR Assay ALDEFLUOR kit (Stem Cell Technologies, Canada) was used to detect the aldehyde dehydrogenase (ALDH) enzymatic activity of the cells. One of them is the ALDEFLUOR® Assay, in which cells with stem cell properties are fluorescently tagged. Materials and Methods Cell lines and cultures An EBV-transformed LCL was established. was determined using a fluorogenic dye-based Aldefluor assay (Stem Cell Technologies, Grenoble, France) ac-cording to the manufacturer’s instructions. Cells were labeled with Aldefluor and analyzed by flow cytometry as described in Materials and Methods. The Aldefluor kit (Stem Cell Technologies, Vancouver, BC, Canada) was used to isolate cell populations with elevated ALDH1 activity, according to the manufacturer's instructions. Max: 15x106, Min: 5x106 4.2 Transfer 3 mL to each of the five (varies based on amount of cells, but minimum amount is 1 mL in one test tube) test tubes (5 ML glass flow tubes) and 1 mL (or less if not available) to tube designated as PI only.
The indicated number of tumor cells was sus-pended in DMEM (50:50 Matrigel) and implanted sub-cutaneously into the flanks of 6-week-old NOD=SCID mice. matic activity was performed using the ALDEFLUOR kit (StemCell Technologies) according to the kit’s protocol. Background We have previously shown that radiotherapy (RT) augments natural killer (NK) functions in pre-clinical models of human and mouse cancers, including sarcomas. Representative flow cytometry analyses of hepatoblastoma cell line HepG2 and HCC cell lines PLC8024 and Hep3B for ALDH activity using the ALDEFLUOR Kit (Stem Cell Technologies). Aldefluor Assay—ALDH enzymatic activity was measured using the Aldefluor kit (Stem Cell Technologies). ALDEFLUOR assay and flow cytometry ALDEFLUOR assay kit was purchased from Stem Cell Technology (#01700) and was performed fol-lowing the manufacturer’sprotocolwithonlyafew modifications. Single cells were isolated from lentivirus-infected primary mammospheres and the Aldefluor kit (Stem Cell Technologies) was used to isolate cells with high ALDH enzymatic activity as described previously .
ALDH1 expression using the Aldefluor kit (Stem Cell Technology, USA), according to the manufacturer’s instructions. aldehyde-oxidizing activity of HSPCs, as measured by Aldefluor stain, is due to Aldh2 and correlates with this protection.
Specialist contract research organaization, offering kits for selection and enrichment from embryonic, epithelial hematopoietic or neural origin, and cloning of either hybridomas or non-adherent stably transfected cells. Flow cytometry and ALDEFLUOR™ assay 5×105–106 cells were stained in round bottom 96 well plates. ALDEFLUOR™ kit for the identification, evaluation and isolation of stem and progenitor cells expressing high levels of ALDH.
The company specializes in developing cell culture media, cell separation products, instruments and other reagents for use in stem cell, immunology, cancer, regenerative medicine and cellular therapy research. ALDEFLUOR assay could be used to select ALDH high and ALDHlow populations to discriminate cancer stem-like cell populations from non-cancer stem-like cells. We assayed the ALDH activity by using the Aldefluor assay kit (Stem Cell Technologies, Inc., Cambridge, MA, USA).
ALDHbright cells are defined as those ALDH+ cells with twice the mean fluorescence intensity (MFI) of the bulk ALDH+ cell population. Storage and Handling: (Read the entire protocol before proceeding) Store kit at –20oC, protect from light.
Briefly, after being stimulated with or without IL-1β, tumor cells were resuspended at a concentration of 1 x 106 cells/ml and incubated with ALDEFLUOR substrate. Briefly, the cells were incubated in the ALDEFLUOR assay buffer containing ALDH substrate BODIPY-aminoacetaldehyde (BAAA), 1 μmol per 1 × 10 6 cells, and incubated for 40 min at 37 °C.
were assayed by Aldefluor assay kit (n = 4).
Dry ALDEFLUOR Reagent is an orange-red powder that changes to a bright yellow-green upon addition of DMSO. Count the cells and adjust cell suspension to a concentration of 1 x 10 6 cells/ml in ALDEFLUOR Assay Buffer. Briefly, 2.5×105 cells were suspended in ALDRFLUOR assay buffer containing ALDH1 substrate and incubated at 37 °C for 45 min. In cell cultures, the activity of this class of enzymes is measured by Aldefluor kit that determines the total activity of ALDH1 family.
Aldefluor assay kit (Aldagen, Durham, NC) was used for the detection of aldehyde dehydrogenase activity according to the manufacturer's instructions. Left, negative controls when the cells were treated with the ALDH inhibitor diethylaminobenzaldehyde (DEAB). Jan Moreb has successfully fractionated human lung cancer cell lines, using the ALDEFLUOR staining method for subsequent in vitro studies. In normal breast tissue, the predominant isoform responsible for RA signaling is ALDH1A116 and it was predicted that, like many other tissues, ALDH1A1 activity was responsible for ALDHbr cells. RALDH activity in individual cells was measured using an Aldefluor kit according to the manufacturer’s protocol. Briefly, DCs were suspended at I x 106 cells/ml in ALDEFLUOR assay buffer containing 5 of activated ALDEFLUOR substrate (150 nM) with or Without the ALDH Inhibitor diethylaminotrnzaldehyde (DEAB) (5 Of 1.5 mM).
Fluorescent substrate of enzymes are used to quantify ALDH activity.
Recently, the ALDEFLUOR assay and a number of ALDH1-specific antibodies have been successfully de-veloped to identify and isolate cancer stem cells in mam-mary gland and breast cancers (9). The EBV-transformed marmoset cell line B95-8 was purchased from Kunming Cell Bank of Chinese Academy of Sciences. Aldefluor fluorescence reveals two cell populations within the SW210.5 and H522 cell lines (left boxes, panels A and B), one bright (R3 region) and the other dim ALDH. It has been proposed that this aberrant population of cells now called “cancer stem cells” (CSCs) drives tumor initiation, progression, metastasis, recurrence, and drug resistance. The CSC population was identified in HeLa cells in terms of measuring ALDH+ cells by use of the ALDEFLUOR™ Kit (STEMCELL Technologies) according to the manufacturer’s instructions and standard operating procedures that are followed at Professor Dubrovska’s lab. Cancer stem cells (CSCs) are cancer cells (found within tumors or hematological cancers) that possess characteristics associated with normal stem cells, specifically the ability to give rise to all cell types found in a particular cancer sample.
In brief, after mixing 5 L of ALDEFLOUR dye with 1 mL of 1 million per mL cell solution in ALDEFLUOR buffer, 0.5 mL of cell solution was trans-ferred to new tube which contained 5 L of ALDH1 specif-ic inhibitor (DEAB, 1.5 mmol/L). ALDEFLUOR assay ALDH activity was analyzed using the ALDEFLUOR™ kit according to manufacturer’s instructions (Stem Cell Technologies, Grenoble, France). The mRNA profile of CSC-related markers was concomitantly observed by quantitative real-time PCR. Proliferation assays were performed to quantify cellular growth over three time points. Both detection strategies indicated that CSC population is below 1% (0.89% +- 0.1% for Aldefluor and 0.71% +- 0.1% for Sox2/Oct4 reporter). Manufacturer: Stemcell Technologies Inc 01700 The ALDEFLUOR™ reagent system is a non-immunological method to identify stem/progenitor cells on the basis of their aldehyde dehydrogenase (ALDH) activity.