A conditionally immortalized human podocyte cell line24 (link) was maintained in RPMI1640 with 10% FBS, Insulin-transferrin-selenium (Gibco), and 50 IU/ml penicillin-streptomycin at 33°C. Podocytes at permissive condition were transfected with plasmids encoding GFP-NCK1 and CD16/CD7/Nephrin cytoplasmic domain (NCD) or CD16/CD7/HA (gift of Dr. Lawrence Holzman, University of Pennsylvania). 24 hrs later, the cells were incubated in RPMI 1640 containing mouse anti-CD16 (Santa Cruz Biotechnology, Inc) at 37°C for 1h and then with in RPMI 1640 containing 1 µg/ml of Alexa Fluor594-conjugated goat anti-mouse IgG (Molecular Probes) for 1h. Then the cells were fixed in 4% paraformaldehyde. Filamentous actin was visualized with Alexa Fluor 350-conjugated phalloidin (Molecular Probes). After mounting, the cells were observed under a confocal microscope. Podocyte expressing CD16/CD7/HA instead of CD16/CD7/NCD or podocyte without crosslink serve as two negative controls to ensure the specificity of the crosslink assay.
Alexa fluor 350 conjugated phalloidin
Manufactured by Thermo Fisher Scientific
Alexa Fluor 350-conjugated phalloidin is a fluorescent probe used for the visualization of F-actin in biological samples. It binds specifically to F-actin and emits fluorescence upon excitation, allowing for the detection and localization of actin filaments in cells or tissues.
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Lab products found in correlation
Insulin transferrin selenium, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 594 conjugated goat anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Mouse anti cd16, by Santa Cruz Biotechnology (2 mentions)
α mem, by Thermo Fisher Scientific (1 mentions)
Cy3 conjugated goat anti rabbit igg h l, by Jackson ImmunoResearch (1 mentions)
Tritc conjugated phalloidin, by Merck Group (1 mentions)
Xfect transfection reagent, by Takara Bio (1 mentions)
Mapple lifeact 7, by Addgene (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit igg h l, by Jackson ImmunoResearch (1 mentions)
3 protocols using alexa fluor 350 conjugated phalloidin
1
Visualization of Podocyte Nephrin Crosslinking
2
Podocyte Crosslinking Assay Protocol
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A conditionally immortalized human podocyte cell line (Saleem et al., 2002 (link)) was maintained in RPMI1640 with 10% FBS, Insulin–transferrin–selenium (Gibco), and 50 IU/ml penicillin–streptomycin at 33 °C. Podocytes at permissive condition were transfected with plasmids encoding GFP-NCK1 and CD16/CD7/Nephrin cytoplasmic domain (NCD) or CD16/CD7/HA (gift of Dr. Lawrence Holzman, University of Pennsylvania). 24 h later, the cells were incubated in RPMI 1640 containing mouse anti-CD16 (Santa Cruz Biotechnology, Inc) at 37 °C for 1 h and then with in RPMI 1640 containing 1 μg/ml of Alexa Fluor594-conjugated goat anti-mouse IgG (Molecular Probes) for 1 h. Then the cells were fixed in 4% paraformaldehyde. Filamentous actin was visualized with Alexa Fluor 350-conjugated phalloidin (Molecular Probes). After mounting, the cells were observed under a confocal microscope. Podocyte expressing CD16/CD7/HA instead of CD16/CD7/NCD or podocyte without crosslink serve as two negative controls to ensure the specificity of the crosslink assay.
3
SV40-transformed WI-38 cell culture and transfection
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WI-38 VA13 cells (JCRB9057; SV40-transformed WI-38 human embryonic lung fibroblasts) were obtained from the Health Science Research Resources Bank (Osaka, Japan). These cells were cultured in MEM-alpha (GIBCO/Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS), 50 U/mL penicillin, and 50 µg/mL streptomycin at 37°C in a humidified incubator containing 5% CO2. pEGFP-NMHC-IIA and pEGFP-NMHC-IIB were kind gifts from Dr. Robert S. Adelstein (NIH, Bethesda, MD). pmCherry-NMHC-IIA was constructed as previously described [18] . mApple-lifeact-7 (plasmid no. 54747) was purchased from Addgene (Cambridge, MA).
Cells were transfected with plasmids using X-fect Transfection Reagent (Takara Bio USA, Mountain View, CA) in antibiotic-free MEM-alpha supplemented with 10% FBS. Rabbit polyclonal anti-NMHC-IIA and anti-NMHC-IIB antibodies targeting the carboxyl termini of NMHC-IIA and NMHC-IIB, respectively, were used as previously described [19, 20] . Alexa Fluor 488-conjugated goat anti-rabbit IgG (H+L) and Cy3-conjugated goat anti-rabbit IgG (H+L) were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). TRITC-conjugated phalloidin was purchased from Sigma-Aldrich (St. Louis, MO). Alexa Fluor 350-conjugated phalloidin was purchased from Molecular Probes (Eugene, OR). Type-I collagen (Cell matrix I-C) was purchased from Nitta Gelatin (Osaka, Japan).
Cells were transfected with plasmids using X-fect Transfection Reagent (Takara Bio USA, Mountain View, CA) in antibiotic-free MEM-alpha supplemented with 10% FBS. Rabbit polyclonal anti-NMHC-IIA and anti-NMHC-IIB antibodies targeting the carboxyl termini of NMHC-IIA and NMHC-IIB, respectively, were used as previously described [19, 20] . Alexa Fluor 488-conjugated goat anti-rabbit IgG (H+L) and Cy3-conjugated goat anti-rabbit IgG (H+L) were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). TRITC-conjugated phalloidin was purchased from Sigma-Aldrich (St. Louis, MO). Alexa Fluor 350-conjugated phalloidin was purchased from Molecular Probes (Eugene, OR). Type-I collagen (Cell matrix I-C) was purchased from Nitta Gelatin (Osaka, Japan).
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