Fluorescein labeling kit nh2

For immunofluorescence assays, cells were suspended in PBS supplemented with 0.5% bovine
serum albumin and 0.05% sodium azide (BSA-PBS). Viable cells ranging in concentration from
1×10⁵ to 1×10⁶ were incubated with fluorescence-labeled monoclonal
antibodies (mAbs), as described below, at 4°C for 1 hr. Stained cells were washed three
times with BSA-PBS and resuspended in BSA-PBS containing propidium iodide (10 µg/mL,
Sigma-Aldrich). Relative immunofluorescence intensities were determined by flow cytometry
using a FACS Canto™ II system (Becton Dickinson, Franklin Lakes, NJ, USA). Anti-CD4 (400×
dilution, CT4), anti-CD8 (400× dilution, CT8), anti-γδ (400× dilution, TCR1), anti-MHC
class II (100× dilution, 2G11), and anti-Bu-1b (200× dilution, 5-11G2) mAbs (all from
Southern Biotech, Birmingham, AL, USA) were used. For fluorescence labeling of mAbs,
Fluorescein Labeling Kit-NH₂, HiLyte™ Fluor 555 Labeling Kit-NH_2,and HiLyte™ Fluor 647 Labeling (F647) Kit-NH₂ (Dojindo Laboratories, Kumamoto,
Japan) were used according to the manufacturer’s instructions.

For fluorescence detection, recombinant proteins (FL, polyG, and Cterm) were labeled with fluorescein using the Fluorescein Labeling Kit-NH₂ according to the manufacturer’s instructions (Dojindo Molecular Technologies Inc.). CGG99 RNA was labeled with CX rhodamine using the Label IT Nucleic Acid Labeling Kit according to the manufacturer’s instructions (Mirus Bio LLC). Recombinant proteins (9 μM; 8 μM nonlabeled proteins and 1 μM fluorescently labeled proteins) were prepared in 10 mM tris-HCl buffer (pH 7.5) containing 25 mM NaCl, 10 mM MgCl₂, and 12% (w/v) glycerol and were then incubated for 1 hour at 23°C. Then, 10% (w/v) 1,6-hexanediol (Sigma-Aldrich) was used to disturb weak interactions that drive LLPS. Fluorescently labeled CGG99 RNA or mTERRA (20 ng/μl) (5′-UUACCGUUACCGUUACCGUUACCG-3′) was prepared in the same buffer. For the preparation of protein/RNA complex solutions, prefolded RNAs with or without 50 μM PpIX were added to the protein solutions, which were then incubated for 1 hour at 23°C. The resultant solutions were covered with coverslips using a 0.12-mm spacer and sealed before measurement. Photobleaching was done with 100% laser power to 50% intensity using the bleaching program of the ZEN software on a Zeiss LSM780 machine. Time-lapse images were recorded every 5 s using a Zeiss Objective Plan-Apochromat 63×/1.4 oil DIC M27 for tracking photorecovery behavior.