Cells were fixed with 4% paraformaldehyde (Nacalai Tesque) in phosphate-buffered saline (PBS), permeabilized with 0.1% Triton X-100 (Nacalai Tesque), and blocked with 20% Blocking One (Nacalai Tesque) in PBST (0.1% Tween-20 in PBS). Antibodies were diluted in 20% Blocking One in PBST (0.1% Tween-20 in PBS). Cells were counterstained with 6-diamidino-2-phenylindole (DAPI; Roche Diagnostics). The following antibodies were used: goat anti-Albumin (ALB; A80-129A, 1:200, Bethyl Laboratories), Rabbit anti-Alpha-fetoprotein (Dako, Glostrup, Denmark), Rabbit anti-MRP2 (JA32-01, Invitrogen), Mouse anti-phospho-Histone H3 (Ser10) Antibody, clone 3H10 (05-806, Millipore), Donkey Anti-Goat IgG (H + L) whole antibody CF 568 Dye (20106, Biotium), Donkey Anti-Goat IgG (H + L) CF657 Dye (20048, Biotium), Donkey Anti-Rabbit IgG (H + L) CF488A (20015, Biotium), and Phalloidin-iFluor™ 488 Conjugate anti-AAT (23115, Bioquest).
Cf488a
Manufactured by Biotium
Sourced in United States
CF488A is a fluorescent dye produced by Biotium. It is a synthetic dye with an excitation maximum of 489 nm and an emission maximum of 505 nm, making it suitable for detection in the green fluorescence channel.
Automatically generated - may contain errors
Lab products found in correlation
Cf633, by Biotium (2 mentions)
Bio gel p 30 gel, by Bio-Rad (2 mentions)
Ab192256, by Abcam (2 mentions)
Ab9661, by Cell Signaling Technology (2 mentions)
Alexa 647, by Thermo Fisher Scientific (2 mentions)
D3571, by Thermo Fisher Scientific (2 mentions)
Prolong gold antifade, by Thermo Fisher Scientific (2 mentions)
Ab15580, by Abcam (2 mentions)
H 3300, by Vector Laboratories (2 mentions)
α actin fitc acta2, by Merck Group (2 mentions)
Irak phosphor t209, by Abcam (2 mentions)
Ab6673, by Abcam (2 mentions)
Cl8942ap, by Abcam (2 mentions)
Gtx109242, by GeneTex (2 mentions)
Pecam 1, by Santa Cruz Biotechnology (2 mentions)
Ab15323, by Abcam (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Lsm 510 meta, by Zeiss (2 mentions)
Lsm 510 meta confocal microscope, by Zeiss (2 mentions)
Paraformaldehyde (pfa), by Nacalai Tesque (1 mentions)
20 protocols using cf488a
1
Immunofluorescence Staining of Hepatocytes
2
Hyaluronic Acid Hydrogel Synthesis
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Hyaluronic acid sodium salt (HA) (700 KDa) was purchased from LifeCore Biomedical (Chaska, MN). 1-heptanol (98%) and NaCl were obtained from Sigma-Aldrich (St. Louis, MO). Dioctyl sulfosuccinate sodium salt (AOT, 96%), divinyl sulfone (DVS, 98%) and, 2,2,4-Trimethylpentane (isooctane, 99%) were purchased from Fisher Scientific (Hampton, NH). CF488A and CF647A amine dye were supplied from Biotium, Inc. (Fremont, CA). N-Hydroxysuccinimide (NHS) was purchased from Thermo Scientific (Rockford, IL). Collagenase type 2 was purchased from Worthington Biochemical (Lakewood, NJ). Erythropoietin (EPO) was purchased from Amgen Inc. (Thousand Oak, CA). CD29 (FITC-conjugated anti-human integrin β1 monoclonal antibody (MAB1951F-100) and CD90 (Alexa Fluor 594-conjugated anti-rat/mouse Thy1.1, Ox-7) were purchased from Millipore (Darmstadt, Germany) and BioLegend (San Diego, CA), respectively.
3
Fluorescent Antibody-based Detection of Protein Aggregates
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Fluorescent antibodies were used for microscopy-based detection of aggregates. The mouse monoclonal anti-α-synuclein Syn211 antibody (Santa Cruz Biotechnology, Inc., Dallas, USA) was labeled with CF633 and CF488A (Biotium, Freemont, USA) and the anti-Aβ IC16 (Heinrich-Heine University, Düsseldorf, Germany) antibody was labeled with CF633 (Biotium, Freemont, USA)40 (link),44 (link). The labeling process was performed according to the manufacturer’s protocol. The dyes with activated succinimidyl esters were mixed with the antibody in carbonate buffer to react covalently with the amines of the antibody. The labeled antibody was purified using a polyacrylamide bead suspension (Bio-Gel P 30 Gel, Bio-Rad Laboratories, Inc., Hercules, USA). The concentration and degree of labeling were determined according to the manufacturer’s protocol.
4
Fluorescent Protein Labeling for Cell Imaging
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Recombinant La 1-375 and BSA were covalently labeled with CF555 or CF488A dyes, respectively, following manufacturer’s instructions (Biotium). Labeled La and BSA were added to fusing RAW 264.7 osteoclasts or C2C12 myoblasts at a final concentration 1.67 μg/ml and incubated at 37 °C for 1 hr. Cells were subsequently washed 4 times with fresh media and fixed for analysis.
5
Reestablishing Protein Corona on Synthetic Nanoparticles
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Ultracentrifugation is the standard method to deplete the corona around synthetic nanoparticles. EVTFF2 preparations were diluted 1:10 in sodium chloride 0.9% buffered with 10 mM HEPES and pelleted via ultracentrifugation at 4°C, 110,000 x g, for 90 min in a Sorval WX80 ultracentrifuge with a TH‐641 rotor at 25,400 rpm all (Thermo Fisher, USA). The resulting pellet was resuspended in the initial sample volume with sodium chloride 0.9% buffered with 10 mM HEPES. To re‐establish a protein corona on the ‘naked’ EVTUCF they were incubated for 1 h at 37°C in EGM‐2 (Lonza) 4% of human serum albumin (Fresenius Kabi, Austria) and VEGF, IGF and EGF (termed VIE/A) (all from Lonza) at dilutions indicated in the figure legend. To re‐establish the protein corona on the naked EVTUCF with fluorescently labelled proteins, BSA‐Alexa flour 488 (BSA‐AF488) (Thermo Fisher, USA), or VEGF165 (R&D Systems, USA) labelled with CF488A (Biotium,USA) according to manufactures protocol, EVs were incubated with 2 mg/ml BSA‐AF488 or 5 μg/ml VEGF‐CF488A for 1 h prior to antibody incubation for visualization of the corona via super resolution microscopy.
6
Immunofluorescence Staining of HELFs for CCN2
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Following the migration/wound healing assay, seeded HELFs were fixed, blocked and stained without being removed from the 24-well plate, using the abovementioned immunofluorescence protocol. As primary and secondary antibodies, anti-CCN2 (Santa Cruz Biotechnology, Dallas, TX, USA) (1:100 dilution) and CF488A (Biotium, Fremont, CA, USA) were used, respectively. Stained cells were simultaneously stained for DAPI and mounted with a non-hardening mounting medium (Ibidi, Gräfelfing, Germany) [33 (link)]. Images were acquired using a 10× air lens (0.45 NA) on a Nikon ECLIPSE Ti2 Inverted Microscope (Nikon, Melville, NY, USA) using NIS-Elements software (Nikon, Melville, NY, USA). Necessary isotype controls were used for this experiment. Confocal images were processed using Fiji software version 2.9.0 [39 (link)].
7
Imaging Chromaffin Cell Exocytosis
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Chromaffin cells were imaged at room temperature using an inverted microscope (Ti-E, Nikon). TIRF microscopy was done using a MLC400 monolithic laser combiner (Agilent Technologies), ZT405/488/561/640rpc dichroic filter (Chroma), and Plan Apo Lambda 100× 1.45 N.A. oil objective (Nikon). Rapid FFN imaging was done using ET455/50 m emission filter (Chroma) and Orca Flash 4.0 C11440-22C sCMOS camera (Hamamatsu). Dual imaging of FFN and BDNF-pHluorin was done using ZET488/561/635m emission filter (Chroma) and iXon DU-897 EMCCD camera (ANDOR). Dual imaging of FFN with Alexa Fluor 488 (ThermoFisher) or CF488A (Biotium) was done using Dual-View filter cube (Optical Insights) mounted with T510lpxrxt beam-splitter (Chroma), ET460/36 m and ET545/40 m emission filters (Chroma), and Orca Flash 4.0 C11440-22C sCMOS camera (Hamamatsu). Confocal imaging of FFN and Alexa Fluor 488 was done using an integrated laser engine (ANDOR), Borealis CSU-W1 spinning disk (ANDOR), ZT405/488/561/640rpcv2 dichroic filter (Chroma), Plan Apo VC 100× 1.4 N.A. oil objective (Nikon), ZET405/488/561/635 emission filter (Chroma), and iXon DU-888 EMCCD camera (ANDOR). Cells were imaged in modified Tyrode's solution, and exocytosis was stimulated with 45 mM K+ by adding an equal volume of stimulation buffer (in mM, 54.5 NaCl, 10 HEPES-NaOH, pH 7.4, 10 glucose, 90 KCl, 5 CaCl2, 1 MgCl2).
8
Recording solution preparation
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Recording solutions contained (mM) 110 NaCl, 2 CaCl2, and 10 HEPES for pH values >6.8. In solutions with a pH of ≤6.8, HEPES was replaced by 10 mM Mes. The pH of all the above-mentioned solutions was adjusted using NaOH. 10-mM stock solutions of Alexa Fluor 488 C-5 Maleimide (AF488; Invitrogen) and CF 488A (Biotium) were prepared in DMSO; aliquots were stored at −20°C.
9
CD8 Antibody Labeling Protocol
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In order to block non-specific Fc-mediated interaction,1 μg of normal Rabbit IgG (ThermoFisher Cat: 31235) is added to the cell sample and incubated for 10 minutes at room temperature. Conventionally, ext., and int. labeled-antibodies are made for staining using the protocols described above (10μg of antibody each); (1) CD8 (clone RPA-T8; Biolegend Cat: 301002) labeled with Atto488 ext. MuSIC probes, (2) CD8 (clone RPA-T8; Biolegend Cat: 301002) labeled with Atto488 int. MuSIC probes, and (3) CD8 (clone RPA-T8; Biolegend Cat: 301002) labeled with CF488A (Biotium Cat: 92446), Antibody concentration is adjusted to 0.25 ug/ul for each sample. The labeled CD8 antibody is added to the cell sample at the appropriate amount as per manufacturer’s recommendations (2μg CD8 antibody / 106 cells) and allowed to incubate in the dark for 20 minutes on ice. Post-incubation, cells are washed twice with 1 ml of cold staining buffer at 300 x g for 5 min. The final cell pellet is resuspended in 0.5 ml of cold staining buffer.
10
Immunostaining and Morphological Analysis of H9C2 Cardiomyoblasts
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H9C2 cardiomyocytes cultured on chamber slides were fixed, permeabilized, and stained using an indirect immunofluorescence method as previously described [34 (link)]. Briefly, cells were incubated with a primary mouse anti-Myosin Heavy Chain (MyHC) antibody (1:100, R&D, Minneapolis, MN, USA) and a goat anti-mouse IgG conjugated to the fluorescent Alexa 488 dye (1:2000, Abcam) secondary antibody, or Phaloidin conjugated with CF 488A (1:2000, Biotium, Fremont CA, USA), and after staining also with DAPI (1 µg/mL; 4083; Cell Signaling), they were viewed under a microscope (Olympus BX40; Olympus Corporation, Tokyo, Japan). In addition, ImageJ software [39 (link)] was used to perform morphological analyses of cardiomyoblasts H9C2 during their differentiation.
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