H1299 cells were detached with 1.5 mM EDTA in PBS −/−, and 1 × 105 cells were counted and transferred to a U-bottom 96-well plate (Sarstedt AG & Co.). To determine the binding of SaroL-1 to surface receptors, cells were incubated with fluorescently labelled protein for 30 min at 4 °C and protected from light, in comparison with PBS-treated cells as a negative control. To deplete cells of glucosylceramide-based glycosphingolipids, they were cultivated 72 h in the presence of 2 μM DL-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP) (Sigma-Aldrich) to inhibit the synthesis of glucosylceramide-based GSLs87 (link) and incubated with fluorescent SaroL-1. Subsequently, cells were centrifuged at 1600 × g for 3 min at 4 °C to remove unbound lectin. The samples were then washed two times with ice-cold FACS buffer (PBS (−/−) supplemented with 3% FCS (v/v)). After the last washing step, the cells were re-suspended with FACS buffer and transferred to FACS tubes (Kisker Biotech GmbH & Co) on ice and protected from light. The fluorescence intensity of treated cells was measured with FACS Gallios from Beckman Coulter. The samples were further analyzed using FlowJo V.10.5.3.
Dl threo 1 phenyl 2 palmitoylamino 3 morpholino 1 propanol ppmp
Manufactured by Merck Group
Sourced in United States
DL-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP) is a synthetic chemical compound. It is a morpholine derivative that inhibits the biosynthesis of glycosphingolipids.
Automatically generated - may contain errors
Lab products found in correlation
U bottom 96 well plate, by Sarstedt (1 mentions)
Facs gallios, by Beckman Coulter (1 mentions)
D luciferin firefly, by Biosynth (1 mentions)
Bovine serum albumin (bsa), by Carl Roth (1 mentions)
Dimethyl sulfoxide (dmso), by Carl Roth (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Carl Roth (1 mentions)
Penicillin streptomycin, by Carl Roth (1 mentions)
Glycerol, by Carl Roth (1 mentions)
2 protocols using dl threo 1 phenyl 2 palmitoylamino 3 morpholino 1 propanol ppmp
1
Analyzing Cellular Glycosphingolipid Binding
2
Comprehensive Immunolabeling Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
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The following antibodies were
used: Alexa Fluor 647-labeled anti-6-His epitope tag (Cat. No. 362611)
from BioLegend (San Diego, CA, USA), Anti-Giantin mouse monoclonal
antibody (Cat. No. ab37266) purchased from Abcam (Waltham, Boston,
USA), LAMP1 (D2D11) XP rabbit monoclonal antibody (Cat. No. 9091)
obtained from Cell Signaling Technology (Danvers, Massachusetts, USA),
and Cy3-AffiniPure F(ab’)2 Fragment Donkey Anti-Rabbit IgG
(H+L) polyclonal antibody (Cat. No. 711-166-152) and Cy3-AffiniPure
Donkey Anti-Mouse IgG (H+L) polyclonal antibody (Cat. No. 715-165-150)
supplied by Jackson ImmunoResearch (West Grove, Pennsylvania, USA).
The following reagents were obtained from commercial sources: PBS,
FBS, HEPES, NEAA, and 0.05% Trypsin-EDTA (1x) were purchased from
Gibco (Thermo Fisher Scientific Inc., Rockford, IL, USA). DMSO, penicillin/streptomycin,
BSA, DAPI, glycerol, methanol, Triton X-100, and sodium hydrogen carbonate
were obtained from Carl Roth GmbH & Co. KG (Karlsruhe, Baden-Württemberg,
Germany).d -Luciferin Firefly was provided by Biosynth (Staad,
Switzerland), and DMEM (with: 1.0 g/L of glucose, stable glutamine,
sodium pyruvate, 3.7 g/L of NaHCO3) was purchased from
PAN Biotech (Aidenbach, Bayern, Germany).dl -Threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol
(PPMP) was obtained from Sigma-Aldrich Chemie GmbH (Saint Louis, MO,
USA).
used: Alexa Fluor 647-labeled anti-6-His epitope tag (Cat. No. 362611)
from BioLegend (San Diego, CA, USA), Anti-Giantin mouse monoclonal
antibody (Cat. No. ab37266) purchased from Abcam (Waltham, Boston,
USA), LAMP1 (D2D11) XP rabbit monoclonal antibody (Cat. No. 9091)
obtained from Cell Signaling Technology (Danvers, Massachusetts, USA),
and Cy3-AffiniPure F(ab’)2 Fragment Donkey Anti-Rabbit IgG
(H+L) polyclonal antibody (Cat. No. 711-166-152) and Cy3-AffiniPure
Donkey Anti-Mouse IgG (H+L) polyclonal antibody (Cat. No. 715-165-150)
supplied by Jackson ImmunoResearch (West Grove, Pennsylvania, USA).
The following reagents were obtained from commercial sources: PBS,
FBS, HEPES, NEAA, and 0.05% Trypsin-EDTA (1x) were purchased from
Gibco (Thermo Fisher Scientific Inc., Rockford, IL, USA). DMSO, penicillin/streptomycin,
BSA, DAPI, glycerol, methanol, Triton X-100, and sodium hydrogen carbonate
were obtained from Carl Roth GmbH & Co. KG (Karlsruhe, Baden-Württemberg,
Germany).
Switzerland), and DMEM (with: 1.0 g/L of glucose, stable glutamine,
sodium pyruvate, 3.7 g/L of NaHCO3) was purchased from
PAN Biotech (Aidenbach, Bayern, Germany).
(PPMP) was obtained from Sigma-Aldrich Chemie GmbH (Saint Louis, MO,
USA).
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