Protocol
Exosome engineering
disease model construction
therapeutic efficacy validation
Exosome engineering
kit method

I.Experimental Procedures
Prepare exosomes
Reagent preparation and storage
ETP preparation: Centrifuge 200 μg of EV-Transit Peptide at 3000-4000 rpm for 1 minute before opening the centrifuge tube cap, then carefully add 1 ml of ETP Dilution Buffer and dissolve thoroughly.
miRNA solution preparation: Add 250 μl of sterile enzyme-free water to 5 nmol of miRNA to dilute it to 20 μM, and dissolve thoroughly.
Nucleic acid loading
Add reagents in the following order and dosage

1. miRNA Solution 2. Exosomes 3. ETP 4. Reaction Buffer
13μl 400μl 53μl 40μl

Incubate at 37°C in the dark for 2 hours with continuous shaking at 150 rpm during the period.
Removal of free small nucleic acids
Transfer the incubated sample to a 100 kDa ultrafiltration tube, make up the volume to 4 ml with Wash Buffer, and centrifuge at 4000×g for approximately 20-30 minutes until the volume is about 200 μl.Add Wash Buffer again to 4 ml for the second washing, and centrifuge at 4000×g until the volume is 200 μl.Gently pipette the exosomes on the filter membrane repeatedly, then transfer them to an empty EP tube.

co-incubation method

During the nucleic acid loading process utilizing the kit, the encapsulation of miRNA molecules within exosomes was performed by Tianjin Jinke Biotechnology Co., Ltd. The experimental protocol was as follows: In the preparation phase, miRNA and exosomes were fluorescently labeled with FAM, Cy5, and PKH26, respectively, for subsequent validation. A co-incubation system was established using a 0.2 cm diameter co-incubation chamber and a citrate-based buffer solution (pH 4.4) consisting of 18.6 mM citric acid and 29.4 mM disodium hydrogen phosphate. For each miRNA loading procedure, a fixed sample volume of 200 μL was maintained, containing both exosomes and miRNA at a molar ratio of 20:1. Following system preparation, the co-incubation chamber was incubated on ice for a minimum of 30 minutes, followed by centrifugation at 160,000 × g for 30 minutes to remove unbound miRNA. The resulting pellet was resuspended in PBS to obtain engineered exosomes loaded with miRNA.

disease model construction
a.in vitro culture of hepatic stellate cells（LX2）

Cell Resuscitation and Culture
1.Rapidly thaw cryopreserved cells in a 37°C water bath, then transfer the cells to a centrifuge tube.
2.Add 5-10 ml of complete medium (DMEM + 15% FBS + 1% triple antibiotics) to the centrifuge tube, and centrifuge at 800 rpm for 5 minutes. After centrifugation, discard the supernatant.
3.Resuspend the cells with medium, then inoculate them into a 100 mm-diameter culture dish.
4.Place the culture dish in an incubator with 37°C and 5% CO₂ for static culture (observe cell status daily and change the medium every two days).
Cell Passage
1.When the cell confluency reaches 80%-90%, discard the old medium and rinse the cells 1-2 times with PBS.
2.Add 3 ml of trypsin to the culture dish, incubate at 37°C for 1-2 minutes, then add 6 ml of complete medium to terminate digestion.
3.Pipette gently to resuspend the cells into a cell suspension, transfer the suspension to a centrifuge tube, and centrifuge at 800 rpm for 5 minutes.
4.Discard the supernatant after centrifugation, resuspend the cells, and passage them at a 1:2 ratio into two 100 mm-diameter culture dishes.
5.Repeat the cell culture steps mentioned above. Continue passaging and culturing the cells until the total number of cells meets the experimental requirements.
Inoculation into 24-Well Plates
1.Add 3 ml of trypsin to each culture dish, incubate at 37°C for 1-2 minutes, then add 6 ml of complete medium to terminate digestion.
2.Pipette gently to resuspend the cells into a cell suspension, transfer the suspension to a centrifuge tube, and centrifuge at 800 rpm for 5 minutes.
3.Discard the supernatant after centrifugation and resuspend the cells.
4.Evenly inoculate the cell suspension into multiple 24-well plates (add 0.5 ml of cell suspension and 0.5 ml of complete medium to each well).
5.Gently shake the 24-well plates to mix the contents, then place them into the incubator.

fibrosis induction

Pathological core induction protocol: Direct stimulation with TGF-β1
1. Cell preparation: Human hepatic stellate cell line LX-2 (retaining key HSC characteristics, high transfection efficiency).
2. Culture conditions: DMEM medium supplemented with 10% fetal bovine serum, at 37°C and 5% CO₂.
3. Inducer: TGF-β1
4. Inducer concentration: 10 ng/mL
5. Induction time: 24 hours, 48 hours, 72 hours (significantly upregulating the expression of α-SMA and Collagen I).
6. Positive control setting: TGF-β1 (10 ng/mL) alone as the induction group, with an untreated group as the control.

therapeutic efficacy validation
Detection of Fibrosis Marker Proteins by Western Blot

Experimental Objective
To quantitatively detect the protein expression level of the activation marker α-SMA in LX-2 cells treated with different exosomes, using GAPDH as an internal control for normalization.

Materials and Reagents
i. Protein samples
ii. 10% resolving gel solution, resolving gel buffer, stacking gel solution, colored stacking gel buffer, modified ammonium persulfate solution, disposable gel casting cups (BOSTER)
iii. Tri-color pre-stained broad molecular weight protein marker (BIOMIKY)
iv. Electrophoresis buffer (1 L: 3.03 g Tris, 14.4 g Glycine, 1.01 g SDS)
v. Transfer buffer (1 L: 3.03 g Tris, 14.4 g Glycine, 20% methanol)
vi. Methanol
vii. Protein-free rapid blocking buffer (BOSTER)
viii. TBST buffer (containing 0.05% Tween-20, washing buffer, 10×) (BOSTER)
ix. Antibody diluent (BOSTER)
x. Primary antibodies: Rabbit anti-human α-SMA monoclonal antibody (Abcam), Mouse anti-human GAPDH monoclonal antibody (Beyotime)
xi. Secondary antibodies: HRP-conjugated goat anti-rabbit IgG (Beyotime), HRP-conjugated goat anti-mouse IgG (Beyotime)
xii. ECL chemiluminescent reagent (BIOMIKY), chemiluminescence imaging system
xiii. PVDF membrane (Roche)
xiv. Shaker
xv. Electrophoresis tank and sandwich-type gel casting glass plates
xvi. Power supply for electrophoresis
xvii. Filter paper, sponge pads

Experimental Methods
SDS-PAGE Gel Preparation and Electrophoresis
i.Preparation of resolving gel (10%): Mix components according to the manufacturer’s instructions. Pour into the glass plate sandwich and overlay with water to level. Allow polymerization for approximately 15 minutes.
ii. Preparation of colored stacking gel: Remove water and blot dry with filter paper. Prepare stacking gel according to the recommended ratios. Fill the remaining space, insert the comb (typically 1.0 mm, 10 wells), and allow polymerization for ~15 minutes.
iii. Sample loading and electrophoresis: Mount the gel in the electrophoresis tank and fill both chambers with electrophoresis buffer. Carefully remove the comb. Load samples from left to right: 6 μL of marker and 35 μL of protein sample per well. Fill unused wells with 4 μL of loading buffer. Change pipette tips between samples. Since the BOSTER colored gel lacks stacking effects, perform electrophoresis at 80 V constant voltage for approximately 100 minutes until the bromophenol blue reaches the gel bottom for optimal results.

Fig 1. Key steps in SDS-PAGE gel preparation and electrophoresis.(A) A researcher performing the gel preparation process. (B) A researcher setting the electrophoresis parameters.

Membrane Transfer
i.Cut the PVDF membrane according to the gel size and activate it by soaking in methanol for 1–2 minutes. Immerse filter paper and sponge pads in transfer buffer.
ii. After electrophoresis, remove the stacking gel and unnecessary portions of the resolving gel.
iii. Assemble the transfer “sandwich” on the transfer cassette in the following order: cathode (black plate) → sponge pad → 3 layers of filter paper → gel → PVDF membrane → 3 layers of filter paper → sponge pad → anode (white plate). Ensure precise alignment and removal of air bubbles between each layer.
iv. Place the cassette into the transfer tank filled with pre-cooled transfer buffer. Keep the apparatus in an ice-water mixture (in a foam box).
v. Perform transfer at a constant current of 300 mA for 60 minutes (α-SMA ~42 kDa; GAPDH ~36 kDa).

Fig 2. A researcher performing the wet transfer process. The transfer cassette is being closed after the assembly of the gel-PVDF membrane stack, which is then placed in a tank filled with pre-chilled transfer buffer and run on ice to prevent overheating.

Immunoblotting and Detection
i.Blocking: After transfer, rinse the PVDF membrane briefly in 1× TBST. Incubate the membrane in protein-free blocking buffer on a shaker at room temperature for 30 minutes.
ii. Primary antibody incubation (α-SMA): Without washing, incubate the blocked membrane with α-SMA primary antibody diluted 1:200 in antibody diluent at room temperature for 2 hours.
iii. Washing: Recover the primary antibody. Wash the membrane with 1× TBST three times, 10 minutes each.
iv. Secondary antibody incubation: Incubate the membrane with HRP-conjugated secondary antibody diluted 1:1000 in antibody diluent at room temperature for 1 hour on a shaker.
v. Washing: Recover the secondary antibody and wash the membrane again three times with 1× TBST, 10 minutes each.
vi. ECL detection: Mix ECL reagents A and B at a 1:1 ratio. Remove excess TBST from the membrane with filter paper. Place the membrane (protein side up) on the imaging plate and evenly add the ECL working solution to cover the entire surface. Incubate for 1–2 minutes, remove excess reagent, and capture the image using a chemiluminescence imaging system. Adjust exposure time to obtain clear, non-saturated α-SMA bands.
vii. Membrane stripping: After imaging, wash the membrane three times with TBST (10 minutes each) and incubate in stripping buffer for 20 minutes.
viii. GAPDH immunoblotting and detection: Perform the same procedure as above. The primary antibody for GAPDH is diluted 1:1000.

Fig 3. Key steps in the immunoblotting process.(A) A researcher performing the antibody incubation process. The PVDF membrane is being incubated with primary antibody solution on a rocking shaker at room temperature to ensure even coverage and facilitate efficient antigen-antibody binding.(B) A researcher performing the membrane washing process.

Data Analysis
Analyze band intensity using ImageJ or similar software. Calculate the grayscale ratio of α-SMA to GAPDH for each sample, and perform statistical analysis to compare the relative expression levels among groups.

LZU Logo
Back to Top