The SiHa and Hela cells (1 × 106 cells/mL), were centrifuged (250×g, 5 min, 4°C); subsequently the supernatant was discarded and then was added 2 mL PBS to wash. The cells were resuspended in 1 mL ALDEFLUOR buffer (STEMCELL Technologies, Vancouver, BC, Canada) and incubated at 37°C for 45 min. 20 μL CD44v6-PE antibody (FAB3660P, R&D Systems, Minneapolis, MN, USA) and 20 μL lgG1-PE, the control, were incubated in a refrigerator at 4°C for 30 min with avoiding light. After centrifuging (250×g for 5 min), and the supernatant was discarded, 500 μL ALDEFLUOR buffer was added to resuspend the cells. The CD44v6(+) cell group and the CD44v6(−) cell group were sorted out with an FCM.
Aldefluor buffer
Manufactured by STEMCELL
Sourced in Canada
The ALDEFLUOR buffer is a solution used in flow cytometry applications. It is designed to maintain the viability and activity of the ALDH enzyme, which is commonly used as a marker for stem and progenitor cells. The buffer helps to preserve the enzymatic function of ALDH, enabling the identification and isolation of ALDH-positive cells.
Automatically generated - may contain errors
Lab products found in correlation
Fab3660p, by R&D Systems (1 mentions)
Aldefluor reagent, by STEMCELL (1 mentions)
Cd133, by Miltenyi Biotec (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Cd105, by BD (1 mentions)
7 aad, by BioLegend (1 mentions)
70 m strainer, by Thermo Fisher Scientific (1 mentions)
Anti h 2kd, by BioLegend (1 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Facsaria, by BD (1 mentions)
Facsaria sorp, by BD (1 mentions)
Aldefluor kit, by STEMCELL (1 mentions)
4 protocols using aldefluor buffer
1
CD44v6 Expression in SiHa and HeLa Cells
2
ALDH Activity and Surface Marker Expression in U-CH1 Cells
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U-CH1 cells were harvested from monolayer by enzymatic dissociation in 0.25% Trypsin and resuspended in 10% FBS in PBS. Cells were resuspended at 1×106 cells/mL in Aldefluor buffer (Stemcell Technologies). 2 µL/mL Aldefluor reagent (Stemcell Technologies) was added to determine aldehyde dehydrogenase (ALDH) activity, with an ALDH inhibitor (diethylaminobenzaldehyde (DEAB)) as a negative control (Stemcell Technologies). Samples were incubated for 30 min, pelleted by centrifugation (470 g, 7 min) and resuspended in Aldefluor buffer (1×106 cells in 200 µL) with fluorochrome-conjugated antibodies specific to human CD90 (555596, BD Biosciences), CD105 (562380, BD Biosciences), CD133 (130090826, Miltenyi Biotec) or IgG controls for 30 min. After incubation, ALDH and cell surface expression was assessed in a minimum of 100,000 cells/treatment group, using a LSR II flow cytometer (BD Biosciences) at the London Regional Flow Cytometry Facility, and analyzed on FlowJo software program. U-CH1 cells were characterized based on side scatter using cluster gating, as previously described [31] (link).
3
Isolation of Mouse PDX Tumor Cells
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PDX-bearing mice were euthanized, and tumors harvested. Tumors were minced and incubated in collagenase (225 U/mL; BioShop Canada Inc., Burlington, ON, Canada) in HBSS, Thermo Fisher Scientific, Mississauga, ON, Canada) at 37 °C on an end-over-end shaker. After 2 h, cell suspension was passed through a 70 µm strainer (Fisher Scientific) and centrifuged for 5 min at 500× g. Cells were resuspended in red blood cell lysis buffer (150 mM NH4Cl, 10 mM KHCO3, 0.1 mM Na2EDTA). After 5 min, cells were centrifuged, resuspended in PBS, and passed through a 70 µm strainer. Cells were centrifuged, resuspended in Aldefluor buffer (Stem Cell Technologies, Inc., Vancouver, BC, Canada), and passed through a 70 µm strainer. Approximately 1 × 107 cells were incubated with anti-H-2Kd (1:1000 SF1-1.1, BioLegend, San Diego, CA, USA) at 37 °C with shaking. After 1 h, cells were centrifuged and resuspended in Aldefluor buffer with 7-AAD (1:10, Biolegend). Stained cells were gated on SSC and FSC to eliminate doublets. 7-AAD- H-2Kd- cells were sorted into ice-cold PBS with 5% BSA (Sigma-Aldrich). H-2Kd purity was assessed (FACS Aria, BD Bioscience, San Jose, CA, USA).
4
ALDH Activity Assay by Flow Cytometry
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We used an ALDEFLUOR kit (STEMCELL Technologies) to detect intracellular ALDH enzymatic activity. The assay was carried out according to the manufacturer's instructions. The activated reagent is converted by intracellular ALDH into the fluorescent product BODIPY‐aminoacetate, which is detectable by flow cytometric analysis. As a negative control, cells were treated with 15 μmol/L diethylaminobenzaldehyde. Cells were incubated for 20 minutes at 37°C in the presence of the above reagents and were stained with fluorescent dye‐conjugated Abs and 7‐AAD in the ALDEFLUOR buffer (STEMCELL Technologies). FACSAria SORP (BD Biosciences) was used for analysis.
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