Aldered aldh detection assay kit

The MKN45, MKN45/ctrl, MKN45/GRP78⁺, and MKN45/sh-GRP78 cells were seeded in six-well plates in quadruplicate at 1 × 10⁵ cells/well for 24 h with or without treatment, washed with cold PBS, and stained with a surface marker antibody for 45 min. After staining, the cells were washed twice with cold PBS before analysis. The expression of cancer stem cell (CSC) markers (CD24, CD44, and LGR5; BD Biosciences, San Jose, CA, USA) on the human gastric cancer cells was analyzed through flow cytometry.
For aldehyde dehydrogenase 1 (ALDH1) analysis, the MKN45 cells were stained using the AldeRed ALDH Detection Assay kit (Sigma-Aldrich, St. Louis, MO, USA) and analyzed through flow cytometry.

After treatment, the cells were washed with cold phosphate buffer saline and stained with surface marker CD24 (BD Biosciences, San Jose, CA, USA) before being analyzed through flow cytometry. For the ALDH1 activity evaluation, the cells were stained using the AldeRed ALDH Detection Assay kit (Sigma–Aldrich, St. Louis, MO, USA) and analyzed through flow cytometry.

For annexin V apoptosis analysis, cells were detached and resuspended in annexin V binding buffer (BD Pharmigen, 51-66121E) and incubated for 15 minutes at room temperature with APC-conjugated AnnexinV (Thermo-eBioscience, BMS306APC-100). After incubation, cells were diluted in binding buffer containing 100 nM of 4’,6-diamidino-2-phenylindole (DAPI). Unstained and single-stained (annexin V and DAPI) were used as gating controls.
For ALDH activity assays, cells were detached, washed in PBS, and stained using the AldeRed ALDH detection assay kit (Merck SRC150) according to manufacturer specifications.
For immunolabeling of CD44 and CD24, cells were detached, washed twice in PBS with 4% FBS, and incubated with CD44-PE (BD Pharmigen, 555479) and CD24-APC (Invitrogen, 17-4714-81) antibodies according to manufacturer specifications at room temperature. After incubations, cells were washed twice in PBS with FBS and resuspended in PBS containing 4% FBS and 100 nM of DAPI. Cells incubated with PE- and APC- conjugated isotype-antibodies and single-stained cells were used as gating controls.
All data were collected on a BD FACS Canto II at the KU Leuven Flow Cytometry Core and analyzed using FlowJo v.10.6.2.

Cells were incubated using either the AldeRed ALDH Detection Assay kit (EMD Millipore, #SCR150) or the ALDEFLUOR kit (STEMCELL Technologies, #C01700) according to the manufacturer’s instructions.

AldeRed ALDH Detection Assay Kit (Merck, #SCR150) was used to detect ALDH activity of cells according to the manufacturer’s instruction. Briefly, cells in 2D culture were treated with 100 ng/ml doxycycline (dox) for 6 days to induce TCOF1 knockout (here LentiV_cas9_puro was used rather than FUCas9cherry), followed by culturing in 3D for 5–6 days. Spheroids were collected and dissociated by trypsin. In all, 2 × 10⁵ cells were then incubated with AldeRed reagent and verapamil for 35 min in 37 °C, protected from light. Cells were centrifuged and pellets were resuspended with 0.5 ml ice-cold ALDE-Red buffer on ice. ALDE-Red signal was detected by Beckman Coulter CytoFLEX S Flow cytometer analyser, using ECM detector (610/20 BP). DEAB was used as a negative control to establish baseline fluorescence.