FITC-PLL

Guidelines (The following recommended experimental protocols are for guidance only and should be adjusted according to your specific requirements)

1. Stock Solution Preparation
1.1 Solvents: Optional solvents include 50% saturated ammonium sulfate; phosphate-buffered saline (PBS, 0.1 M, pH 7.4); 100 mM NaCl solution.
1.2 Recommended Concentrations:
1.2.1 Prepared via the reaction of FITC (Isomer I) with poly-D-lysine polymer under alkaline conditions, using different FITC:lysine molar ratios (e.g., 1:51, 1:31, 1:13 for PDL1; 1:459, 1:92, 1:46 for PDL2).
1.2.2 100 μg/mL; 1-10 mM.
1.2.3 Prepared by dispersing 1 mg PLL-StNP in 1 mL PBS buffer (pH 7.4), followed by the addition of 0.05 mg FITC.

2. Working Solution Preparation
2.1 Diluents: Optional diluents include DMEM; 100 mM NaCl solution; serum-free DMEM medium.
2.2 Working Concentrations:
2.2.1 5-200 µg/mL.
2.2.2 Adjust to the target molar ratio of amino acid residues to charged lipid molecules (Rₚ), while maintaining the final lipid concentration at 100 μM.
2.3 Notes: Adjust the working solution concentration as needed; prepare and use immediately.

3. Staining Procedures
3.1 Sample Type Description
3.1.1 Adherent and Suspension Cell Lines^[1][4]:
3.1.1.1 Adherent cells: Mouse macrophage cell line J774, HeLa cells; cultured on coverslips for 12-36 h before staining; no trypsin digestion required prior to staining.
3.1.1.2 Suspension cells: Mouse myeloma B cell line SP2/0; no centrifugation required during staining.
3.1.2 Glass coverslip surfaces: Substrates for subsequent neuronal cell culture.
3.1.3 Phospholipid vesicles: Large unilamellar vesicles (LUVs) prepared from POPG, DPPG, POPG/POPC (1/1 mol/mol) or DPPG/DPPC (1/1 mol/mol).
3.2 Incubation Conditions
3.2.1 Adherent and Suspension Cell Lines:
3.2.1.1 Incubate with FITC-PDL working solution at 37°C for 10-30 min; for long-term imaging, the incubation time is 10 h.
3.2.1.2 Incubate HeLa cells with 80 μg/mL FITC-PLL-StNP-200 working solution for 2 h; then continue incubation in fresh DMEM containing 10% fetal bovine serum for another 3 h; for lysosome colocalization, incubate with a lysosomal red fluorescent probe for 5-10 min after the completion of FITC-PLL-StNP-200 incubation.
3.2.2 Glass Coverslip Surfaces:
3.2.2.1 Binary patterned lines: When used in combination with LN to form binary lines, first allow LN to diffuse through side channels for 5-10 min, then flush the channels with FITC-PLL to apply an adhesion layer.
3.2.2.2 Diffusion-mediated patterning: Allow FITC-PLL to diffuse through microfluidic interconnected channels to form a surface gradient; complete permeation can be achieved within 40 min, with the adsorption range consistent with the coverage of the source channel.
3.2.3 Phospholipid Vesicles: Incubate at 20-23°C with continuous stirring; use FITC-PLL working solution adjusted to the target Rₚ value (0.4-2).
3.3 Washing Procedures
3.3.1 Adherent and Suspension Cell Lines: Wash with DMEM after incubation; rinse cells with PBS after incubation.
3.3.2 Glass Coverslip Surfaces:
3.3.2.1 Binary patterned lines: Aspirate the FITC-PLL solution, thoroughly rinse the channels and coverslips with sterile deionized water, then aspirate the liquid until dry.
3.3.2.2 Diffusion-mediated patterning: Flush the channels with FITC-PLL after LN incubation to add an adhesion layer to the substrate pattern.
3.3.3 Phospholipid Vesicles: No washing step is specified.

4. Control Setup
4.1 Negative Control: Incubate cells without adding the FITC-PDL probe to detect autofluorescence.
4.2 Inhibition Control: Pre-treat cells with 3 mM Amiloride (HY-B0285) for 15 min before FITC-PDL incubation; perform intracellular K⁺ depletion on cells before FITC-PDL incubation.
4.3 Competition Control: Incubate cells with FITC-PDL and excess unlabeled poly-D-lysine simultaneously; incubate cells with FITC-PDL and competitive monovalent ligands (e.g., L-Phenylalanine (HY-N0215), 2-phenyloxazolone) simultaneously.

5. Detection and Analysis
5.1 Instrument Types: Flow cytometer; two-photon fluorescence microscope; fluorescence microscope; fluorescence spectrophotometer; inverted fluorescence microscope.
5.2 Excitation/Emission Wavelengths:
5.2.1 Flow cytometer: ~488 nm excitation, ~525 nm emission.
5.2.2 Two-photon fluorescence microscope: 960 nm excitation (equivalent to 480 nm in single-photon excitation), with emission light in the blue-green region.
5.2.3 Fluorescence spectrophotometer: 490 nm excitation, 520 nm emission.
5.2.4 Inverted fluorescence microscope: 495 nm excitation, 524 nm emission.
5.2.5 Fluorescence microscope: Wavelengths compatible with FITC.
5.3 Result Analysis
5.3.1 Cell Line Samples:
5.3.1.1 Changes in fluorescence intensity: The mean fluorescence intensity increases linearly with the rise of FITC epitope density until each polymer contains approximately 16 FITC groups; the fluorescence intensity decreases when the density increases further; probes with high-density substitution show significant fluorescence quenching in acidic vacuoles; FITC-PLL-StNP-200 exhibits stable bright green fluorescence.
5.3.1.2 Fluorescence localization: FITC-PDL localizes to acidic endocytic vacuoles (endosomes) in macrophages; the green fluorescence of FITC-PLL-StNP-200 colocalizes with the red fluorescence of lysosomal probes in HeLa cells, indicating its localization in lysosomes; merged images of colocalized regions show yellow fluorescence.
5.3.1.3 Fluorescence lifetime: The average lifetime is approximately 2.0 ns in acidic intracellular compartments, and approximately 3.7-3.9 ns under neutral pH conditions.
5.3.1.4 Kinetic analysis: Calculate the receptor occupancy fraction and apparent binding rate constant to quantify binding efficiency.
5.3.2 Glass Coverslip Surface Samples:
5.3.2.1 Fluorescence localization: FITC-PLL forms bound patterns or gradients on the surface of glass coverslips.
5.3.2.2 Functional results: FITC-PLL can act as a cell adhesion promoter to support neuronal attachment, while enabling visualization of treated channel structures to evaluate neuronal growth direction; when used in combination with LN gradients, it can restrict neuronal protrusions to narrow regions or guide axonal directional growth.
5.3.3 Phospholipid Vesicle Samples:
5.3.3.1 Changes in fluorescence intensity: Monitor changes in normalized fluorescence intensity over time; a significant decrease in intensity indicates that FITC-PLL binds to and penetrates the lipid membrane.
5.3.3.2 After binding to all tested membranes, the normalized fluorescence intensity value of short-chain FITC-PLL (with an average of 106 amino acid residues) is ≤0.09, indicating its ability to penetrate both fluid-phase and gel-phase membranes.
5.3.3.3 After binding to all tested membranes, the normalized fluorescence intensity value of long-chain FITC-PLL (with an average of 319 amino acid residues) ranges from 0.46 to 0.63, indicating its limited penetration ability.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.