NF90–8 cells in 6-well plates were incubated with AAV-DJ control, AAV-DJ-GRD-2HA or AAV-DJ-GRD-C10–2HA at MOI 5000 for 36 hrs. Cells were trypsinized, fixated by the IC fixation buffer (Invitrogen, #00–8222), permeabilized by the permeabilization buffer (Invitrogen, #00–8333) and incubated with anti-HA antibody conjugated with AlexaFluor 647 (R&D Systems, #IC6875R) at 5 μl/ 106 cells in 100 μl permeabilization buffer for 45 min at RT. Cells were then washed twice in 2 ml permeabilization buffer, resuspended in 0.5 ml flow buffer and analyzed by a BD FACSAria Fusion. Data were obtained from three independent samples and analyzed by two-tailed t-test.
Alexafluor 647
Manufactured by R&D Systems
AlexaFluor 647 is a fluorescent dye used for labeling and detection applications in biological research. It has an excitation maximum at 650 nm and an emission maximum at 665 nm, making it suitable for use with red-light excitation sources and detection in the far-red region of the spectrum.
Automatically generated - may contain errors
Lab products found in correlation
Permeabilization buffer, by Thermo Fisher Scientific (2 mentions)
Facsaria fusion, by BD (2 mentions)
Ic fixation buffer, by Thermo Fisher Scientific (2 mentions)
Permeabilization buffer, by BD (2 mentions)
Permeabilization buffer, by R&D Systems (2 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Sylgard 184 elastomer, by Dow (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Ingenuity pathway analysis (ipa), by Merck Group (1 mentions)
Ab92547, by R&D Systems (1 mentions)
Ip l 780, by Nanoscribe (1 mentions)
4 protocols using alexafluor 647
1
Quantification of HA-tagged Protein Expression
2
Quantification of HA-tagged Protein Expression
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NF90–8 cells in 6-well plates were incubated with AAV-DJ control, AAV-DJ-GRD-2HA or AAV-DJ-GRD-C10–2HA at MOI 5000 for 36 hrs. Cells were trypsinized, fixated by the IC fixation buffer (Invitrogen, #00–8222), permeabilized by the permeabilization buffer (Invitrogen, #00–8333) and incubated with anti-HA antibody conjugated with AlexaFluor 647 (R&D Systems, #IC6875R) at 5 μl/ 106 cells in 100 μl permeabilization buffer for 45 min at RT. Cells were then washed twice in 2 ml permeabilization buffer, resuspended in 0.5 ml flow buffer and analyzed by a BD FACSAria Fusion. Data were obtained from three independent samples and analyzed by two-tailed t-test.
3
Immunostaining of NF-κB Transcription Factor
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Antibodies against the Myc tag on NF‐κB p65/RelA (D14E12) XP were purchased from Cell Signaling Technologies. Human/mouse anti‐RelA/NF‐κB p65 and the rat IgG2A isotype control (54447) [Alexa Fluor® 647] antibodies were purchased from R&D Systems. The His‐tagged mAb was purchased from Novagen. The donkey anti‐sheep IgG H&L (Alexa Fluor® 488) (ab150177) antibody was purchased from Abcam. The Alexa Fluor 514‐conjugated goat anti‐mouse IgG (H + L) cross‐adsorbed secondary antibody and the Alexa Fluor 647‐conjugated goat anti‐rabbit IgG (H + L) cross‐adsorbed secondary antibody were purchased from Thermo Fisher Scientific. For confocal microscopy, anti‐tag antibodies were used at a final dilution of 1:100, the human/mouse anti‐RelA/NF‐κB p65 antibody was used at a final dilution of 1:20, and all of the secondary antibodies were used at a 1:500 dilution.
4
Photocrosslinkable Resin for 3D Microstructures
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Photocrosslinkable resin IP-L 780 (Nanoscribe GmbH) was used for producing master 3D microstructures by 2PP. The structures were developed using propylene glycol monomethyl ether acetate (PGMEA, RER600, Arch Chemicals) and rinsed using isopropyl alcohol (IPA, Sigma-Aldrich). Polydimethylsiloxane (PDMS, Sylgard 184 elastomer, Dow Corning) and fluoroctatrichlorosilane (FOTS, Sigma-Aldrich) were used for microlithography procedure to finally emboss PLA films (Corbion Purac®, Corbion), providing them with microstructures. Components for cell culture such as Minimum Essential Medium α (α-MEM), fetal bovine serum (FBS), penicillin-streptomycin (Pen/Strep), L-glutamine (L-glu) and trypsin in ethylenediaminetetraacetic acid (trypsin-EDTA) were from Gibco, Life Technologies. Bovine serum albumin (BSA) was from Sigma-Aldrich. For fluorescence microscopy, the following probes were used: fluorescein isothiocyanate (FITC) conjugated antibody for vinculin (F7053, Sigma-Aldrich), antivimentin (ab92547) primary antibody and anti-rabbit Alexa Fluor® 647 as secondary antibody (from R&D Systems), CF™594 phalloidin (Biotium) for cytoskeleton, 4′,6-Diamidino-2-phenylindole dihydrochloride for nuclei (DAPI, Sigma-Aldrich).
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