Human HAP1 cells (Horizon Discovery, #C631) were maintained in Iscove’s Modified Dulbecco’s Medium (IMDM) supplemented with 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin (Invitrogen). Cells were cultured in a humidified incubator at 37 °C with 5% CO2. Transfection of plasmids into HAP1 cells was carried out with Lipofectamine 3000 (Life Technologies) following manufacturer’s instructions. Cell morphology was evaluated with phase contrast images taken at 20x with the EVOS FLc system (Life Technologies). Visible vacuoles (>0.4 um) were measured using ImageJ software [33 (link)]. Ratiometric measurement of lysosomal pH was carried with the dye Oregon Green 488-dextran as previously described [21 (link)]. LAMP2 antibody # H4B4 was obtained from the Developmental Studies Hybridoma Bank, University of Iowa, and used at 3 ug/ml. The secondary antibody was anti-Mouse IgG Alexa fluor plus 488 from Invitrogen, A32723, used at 1/2000 dilution.
Anti mouse igg alexa fluor plus 488
Manufactured by Thermo Fisher Scientific
The Anti-Mouse IgG Alexa Fluor Plus 488 is a fluorescent-labeled secondary antibody used for detection and visualization in immunological applications. It specifically binds to mouse immunoglobulin G (IgG) antibodies and emits green fluorescence when excited by light at the appropriate wavelength.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Evos flc system, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Hap1 cells, by Horizon Discovery (1 mentions)
Mouse anti myc monoclonal antibody, by Santa Cruz Biotechnology (1 mentions)
Lsm 700 confocal laser scanning microscope, by Zeiss (1 mentions)
Zen software, by Zeiss (1 mentions)
2 protocols using anti mouse igg alexa fluor plus 488
1
Characterization of Lysosomes in HAP1 Cells
2
Immunolocalization of Transgenic Protein
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Transgenic hairy roots carrying the LjUBQpro:LjNLP1-myc construct were fixed in 3% paraformaldehyde in MTSB buffer (50 mM PIPES-KOH (pH 7.0), 5 mM EGTA, 5 mM MgSO4) for 40 min under vacuum. After washing with wash buffer (MTSB buffer with 0.1% Triton X-100), roots were preincubated in 3% BSA in wash buffer at room temperature for 1 h and then incubated with a 1:100 dilution of an anti-myc mouse monoclonal antibody (Santa Cruz Biotechnology Inc.) in 3% BSA in wash buffer at room temperature overnight. After washing with wash buffer, the roots were incubated with a 1:1,000 dilution of anti-mouse IgG-Alexa Fluor Plus 488 (Invitrogen) in 3% BSA in wash buffer at room temperature for 3 h. Before observing the signal, the roots were stained with 5 μg mL−1 4ʹ, 6-diamidino-2-phenylindole (Dojindo) for 15 min. Fluorescent images were obtained using a LSM700 confocal laser-scanning microscope (Carl Zeiss) equipped with ZEN software (Carl Zeiss).
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