Human follicular DP cells were purchased from PromoCell (Germany) and cultured in Follicle DP Cell Growth Medium kit (PromoCell) at 37°C in a humidified atmosphere of 5% CO2 in an incubator. The medium was changed every 3 days, and the cells were harvested with Accutase cell detachment solution purchased from Merck Millipore (USA). Additionally, 293T cells were purchased from American Type Culture Collection (USA). Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher Scientific, USA) was supplemented with 5% (v/v) fetal bovine serum (FBS) purchased from Sigma-Aldrich, USA. LY294002, a selective phosphatidylinositol 3-kinase (PI3K) inhibitor, and ginsenoside Rg4, derived from black ginseng, were purchased from Merck (Germany) and AREZ Co. Ltd., (Korea), respectively. The T cell-specific transcription factor and lymphoid enhancer-binding factor (TCF/LEF) luciferase reporter plasmids were purchased from Promega (USA). Minoxidil was purchased from Sigma-Aldrich.
Accutase cell detachment solution
Manufactured by Merck Group
Sourced in United States, Germany
Accutase cell detachment solution is a proteolytic enzyme mixture specifically formulated for the gentle dissociation of adherent cells from cell culture surfaces. It is designed to effectively detach cells while minimizing cell damage or disruption of cell surface proteins.
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Lab products found in correlation
Facsaria cell sorter, by BD (3 mentions)
Alexa fluor 647 conjugated cholera toxin b subunit, by Thermo Fisher Scientific (3 mentions)
Alexa fluor 488 conjugated secondary antibody, by Thermo Fisher Scientific (3 mentions)
Dexamethasone (dex), by Merck Group (3 mentions)
Gelatin, by Merck Group (3 mentions)
Lsrii flow cytometer, by BD (3 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Collagenase 4, by Thermo Fisher Scientific (2 mentions)
Egm medium, by Lonza (2 mentions)
Essential medium α, by Thermo Fisher Scientific (2 mentions)
Dnase 1, by Thermo Fisher Scientific (2 mentions)
Hank s balanced salt solution, by Lonza (2 mentions)
Pen strep, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Vitrocell (2 mentions)
Neurobasal medium, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
B27 supplement, by Thermo Fisher Scientific (2 mentions)
51 protocols using accutase cell detachment solution
1
Human Follicular Papilla Cell Culture
2
CD133 Expression Analysis in Cells
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Cells were harvested and dissociated as previously described16 , 17 using Accutase cell detachment solution (Merck Millipore). Dissociated single cells were incubated with rabbit polyclonal anti‐human CD133 (ab19898; Abcam) diluted in FACS buffer (0.5% [w/v] BSA and 0.1% [w/v] sodium azide in PBS) for 30 minutes on ice. Cell suspensions were then washed and incubated with Alexa Fluor 488‐conjugated secondary antibody (Molecular Probes) diluted in FACS buffer for 30 minutes on ice. Cells incubated with secondary antibody only served as negative controls. Cells were sorted using a FACSAria Cell Sorter (Becton Dickinson) and mean fluorescence intensities (MFI) were calculated by subtracting the intensity of the negative controls.
3
Antibody Staining and FACS Analysis
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As previously described (Sasaki et al., 2015 (link)), cells were harvested with Accutase® cell detachment solution (Merck Millipore, Billerica, MA, United States), and dissociated single cells were incubated with primary antibodies diluted in fluorescent activated cell sorting (FACS) buffer (0.5% [w/v] bovine serum albumin [BSA] and 0.1% [w/v] sodium azide in PBS) for 30 min on ice. After washing, the cell suspension was incubated with Alexa Fluor® 488-conjugated secondary antibodies (Molecular Probes, Eugene, OR, United States) and diluted in FACS buffer for 30 min on ice. For the detection of GM1 or c-series gangliosides, cells were incubated with Alexa Fluor® 647-conjugated cholera toxin B subunit (Molecular Probes) or APC-conjugated anti-A2B5 antibody (Miltenyi Biotec, Bergisch Gladbach, Germany) diluted in FACS buffer for 30 min on ice, respectively. Cell sorting and analysis were performed using a FACSAria™ Cell Sorter. The following primary antibodies were used: anti-GM3 (NBT Laboratories Inc, Tokyo, Japan), anti-GM2 (TCI, Tokyo, Japan), anti-GD1a (TCI), anti-GT1b (TCI), anti-GD3 (Merck Millipore), anti-GD2 (TCI), and anti-GD1b (TCI). Mean fluorescence intensity was calculated by subtracting the intensity of the control.
4
Dexamethasone Modulation of Glioblastoma Stem Cells
5
Cell Surface ICAM-1 and GM1 Expression
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As described in our previous reports [8 (link), 9 (link)], cells were harvested with the Accutase® cell detachment solution (Merck Millipore, Billerica, MA, USA) and dissociated single cells were incubated with FITC-conjugated ICAM-1 (BioLegend, San Diego, CA, USA) or FITC-isotype control (Becton Dickinson, Franklin Lakes, NJ, USA) diluted in FACS buffer (0.5% [w/v] BSA and 0.1% [w/v] sodium azide in PBS) for 30 min on ice. To detect GM1, cells were incubated with Alexa Fluor® 647-conjugated cholera toxin B subunit (Molecular Probes, Eugene, OR, USA) diluted in FACS buffer for 30 min on ice. After washing, cell sorting and analysis were performed using a FACSAria™ Cell Sorter (Becton Dickinson). Mean fluorescence intensities (MFIs) were calculated by subtracting the intensities of the controls.
6
Spheroid Dissociation and Lipid Droplet Analysis
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For dissociation of spheroids to obtain single spheroid-derived cells, five spheroids were pooled and washed with PBS. One hundred fifty microliter Accutase cell detachment solution (Merck Millipore, #SCR005) was added, followed by careful pipetting. The spheroid solution was incubated for 10 min in Accutase solution followed by another round of pipetting, resulting in a cell suspension.
For LD analysis, the obtained cell suspension was seeded into glass-bottom cellview cell culture dishes (Greiner bio one, #627965) and allowed to re-attach for 4 h. The cells were washed with PBS and incubated in 1:1000 BODIPY 493/503 (Invitrogen, #D3922) in culture medium for 10 min at 37 °C. Cells were washed twice with warm PBS and fixed by incubation with warm 3.7% formaldehyde solution for 10 min at 37 °C. Cells were washed again and mounted in 25 μl Vectashield Antifade Mounting Medium with DAPI (Vector Laboratories, #H-1200-10). LD imaging and volume analysis was performed as described previously (19 (link)). Briefly, images were acquired on a Nikon A1+ confocal laser scanning microscope and analyzed with ImageJ. LD volume was quantified and normalized to cell count. Average LD volume per cell was calculated from three ATGL-KO clones and three control clones from a total of 76 or 80 cells, respectively.
For LD analysis, the obtained cell suspension was seeded into glass-bottom cellview cell culture dishes (Greiner bio one, #627965) and allowed to re-attach for 4 h. The cells were washed with PBS and incubated in 1:1000 BODIPY 493/503 (Invitrogen, #D3922) in culture medium for 10 min at 37 °C. Cells were washed twice with warm PBS and fixed by incubation with warm 3.7% formaldehyde solution for 10 min at 37 °C. Cells were washed again and mounted in 25 μl Vectashield Antifade Mounting Medium with DAPI (Vector Laboratories, #H-1200-10). LD imaging and volume analysis was performed as described previously (19 (link)). Briefly, images were acquired on a Nikon A1+ confocal laser scanning microscope and analyzed with ImageJ. LD volume was quantified and normalized to cell count. Average LD volume per cell was calculated from three ATGL-KO clones and three control clones from a total of 76 or 80 cells, respectively.
7
Robust GBM cell line isolation and culture
8
Investigating Cannabinoid Receptor Signaling
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CBD was bought from Biomol GmbH (Hamburg, Germany). JWH-133 and THC were bought from Bio-Techne GmbH (Wiesbaden, Germany) and Lipomed GmbH (Herne, Germany), respectively. R(+)-methanandamide, capsazepine and NAC were obtained from Sigma-Aldrich (Taufkirchen, Germany). AM251 and AM630 were obtained from Biozol (Eching, Germany). O-1602 and O-1918 were purchased from Tocris Bioscience (Bristol, UK) and Cayman Chemicals (Ann Arbor, Michigan, USA), respectively. SnPPIX was obtained from Enzo Life Sciences (Lörrach, Germany). The transfection reagent Lipofectamine™ RNAiMAX, OptiMEM and hPDGF-BB were purchased from Thermo Fisher Scientific Inc. (Schwerte, Germany). siRNA targeting HO-1 was purchased from Santa Cruz Biotechnology (Heidelberg, Germany; sc-35554). Negative control siRNA was from Qiagen (Hilden, Germany; cat. no. 1022076). Accutase cell detachment solution was obtained from Merck Chemicals GmbH (Darmstadt, Germany).
9
Cytogenetic Analysis of GRX Cells
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GRX cells were detached from the petri dish by Accutase cell detachment solution (Merck, Sigma-Aldrich, Taufkirchen, Germany) and transferred in T25 flasks and cultured for two days. The cultures were harvested following standard protocols including treatment with colcemid (10 µg/mL), hypotonic KCl solution (0.4%), and fixative (methanol: acetic acid (3:1)). G-banding by pancreatin staining with Giemsa (GPG) was performed following standard procedures. Cytogenetic analysis was done using an Axioplan 2 imaging microscope (Carl Zeiss, Oberkochen, Germany). For conventional chromosome analysis, five cells were analyzed by light microscopy. Karyotyping was done by using the IKAROS karyotyping system, version 5.9.1 (MetaSystems, Altlussheim, Germany). With this method, an average banding resolution of 250 bands was achieved. Aberrations were described according to the guidelines proposed by the International Committee on Standardized Genetic Nomenclature for Mice that were revised in September 2021 [22 ].
10
Immunophenotyping of Cell Surface Antigens
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In general, trypsinization reduces the abundance of some cell surface antigens. Therefore, to avoid this effect, cells were harvested with Accutase® cell detachment solution (Merck Millipore, Billerica, MA), and these dissociated single cells were incubated with primary antibodies diluted in FACS buffer (0.5% (w/w) BSA and 0.1% (w/w) sodium azide in PBS) for 30 min on ice. After washing, the cell suspension was incubated with Alexa Fluor® 488- or Alexa Fluor® 647-conjugated secondary antibodies (Molecular Probes, Eugene, OR) diluted in FACS buffer for 30 min on ice. For the detection of GM1, cells were incubated with Alexa Fluor® 647-conjugated cholera toxin B subunit (Molecular Probes) diluted in FACS buffer (0.5% BSA and 0.1% sodium azide in PBS) for 30 min on ice. Cell sorting and analysis were performed using a FACSAriaTM Cell Sorter (BD Biosciences). We used the following primary antibodies: anti-GM3 (NBT Laboratories Inc., Tokyo, Japan); anti-monosialotrihexosylceramide (GM2) (TCI, Tokyo, Japan); anti-monosialotrihexosylceramide (GD1a) (TCI); anti-ganglioside GD3 (Merck Millipore); anti-ganglioside GD2 (TCI); anti-ganglioside GD1b (TCI); and anti-insulin receptor (IR)α (Abcam, Cambridge, UK).
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