Reagents List
|
Product Name |
Cat.NO. |
|
40801ES |
THP-1-DNA Transfection Protocol
Cell Culture and Passaging (T25 Flask)
Cell Counting: Remove the cell culture from the incubator. Gently resuspend the cells by pipetting to ensure a homogeneous suspension. Assess cell viability and density using an automated cell counter.
Cell Passaging(Direct Dilution Method): Based on the cell count, dilute the culture to approximately 5 × 10⁵ cells/mL with fresh complete medium for passaging.
Cell Passaging(Half-Medium Replacement Method): If the culture supernatant appears yellow (indicating acidification), carefully remove the plate from the incubator and keep it undisturbed to allow cells to settle. Tilt the plate gently so that cells sediment at the bottom, then aspirate the old supernatant using a pipette. Replace with fresh complete medium, mix gently, and proceed with counting and passaging.
Cell Passaging(Centrifugation-Based Passaging): If cell health is suboptimal, passage cells via low-speed centrifugation: transfer the cell suspension to a sterile tube and centrifuge at 200 × g for 3–5 minutes. Discard the supernatant, resuspend the pellet in fresh complete medium, and perform cell counting before reseeding.
[Note]: When healthy, these cells typically grow in grape-like clusters.
Cell Transfection (24-Well Plate)
I. Preparation
1. Cell Preparation: 24 hours before transfection, collect the cell suspension and perform cell counting. Seed 1 × 10⁵ cells (100,000 cells) per well into a 24-well plate, adding 500 μL of complete growth medium per well (total medium volume)
2. Reagent Preparation:
- Plasmid DNA: Use high-purity plasmid DNA (e.g., purified by kit), ideally at a concentration >1000 ng/μL.
- Transfection Reagent: Allow Hieff Trans™ Booster Transfection Reagent to warm to room temperature. Gently mix before use.
- Serum-Free Medium: Prepare serum- and antibiotic-free basal medium (e.g., Opti-MEM) for diluting DNA and transfection reagent.
II. Complex Formation
1. Dilute DNA: In a sterile microcentrifuge tube, add 25 μL serum-free medium followed by 0.5 μg plasmid DNA. Mix gently. Then add 1 μL Enhancer, and mix again to obtain diluted DNA.
2. Dilute Transfection Reagent: In another sterile tube, add 25 μL serum-free medium and 1 μL Hieff Trans™ Booster Transfection Reagent. Mix gently.
3. Combine and Incubate: Add the diluted DNA solution to the diluted transfection reagent tube. Mix gently by pipetting. Incubate at room temperature for 10–15 minutes to form DNA-transfection reagent complexes.
III. Transfection
1. Medium Change: Before transfection, carefully aspirate the old medium and replace with 0.5 mL pre-warmed complete medium.
2. Add Complexes: Add the incubated DNA-transfection reagent complexes dropwise and evenly to the cells. Gently rock the plate to distribute.
3. Incubation: Return cells to a 37°C, 5% CO₂ incubator for culture.
IV. Post-Transfection Handling
1. Medium Replacement: 4–6 hours post-transfection, check cell morphology. If significant toxicity is observed, aspirate the medium containing complexes and replace with 1 mL fresh pre-warmed complete medium. If cells appear healthy, medium change is optional.
2. Analysis: After 24–72 hours, assess transfection efficiency or functional outcomes based on experimental design (e.g., fluorescence observation, mRNA or protein detection).
Tips:
1. If significant cytotoxicity occurs, replace medium after 6 h or reduce Enhancer volume by half to mitigate toxicity.
2. If transfection efficiency is low, increasing the amount of transfection reagent may improve results.
3. For optimal performance, dilute DNA and transfection reagent in Opti-MEM rather than DMEM.
4. The transfection system is compatible with serum and antibiotics; however, DNA and reagent dilutions must be performed in serum- and antibiotic-free medium.
Experimental Results Analysis
Plasmid DNA was transfected into THP-1 human monocytic leukemia cells using Yeasen Booster Transfection Reagent and a leading imported reagent (Brand T3000) in parallel. Transfection efficiency was assessed by qPCR 24 hours post-transfection.
Figure 1. Plasmid DNA transfection in THP-1 cells using Yeasen Booster transfection reagent, demonstrating high transfection efficiency.
Different Cell Culture Vessel Transfection Volumes (for reference only):
|
Culture vessel |
Medium Volume |
DNA Transfection |
siRNA Transfection (Final Concentration 50 nM) |
||||
|
Volume of Medium
|
Volume of Opti-MEM Complex
|
DNA(μg) |
Booster Transfection Reagent (μL) |
Transfection Enhancer (μL) |
Volume of siRNA (Initial Concentration 20 μM) |
Booster Transfection Reagent (μL) |
|
|
96-well |
100 μL |
2×5 μL |
0.1 |
0.2 |
0.2 |
0.25 μL |
0.3 |
|
48-well |
250 μL |
2×12.5 μL |
0.25 |
0.5 |
0.5 |
0.625 μL |
0.75 |
|
24-well |
500 μL |
2×25 μL |
0.5 |
1 |
1 |
1.25 μL |
1.5 |
|
12-well |
1 mL |
2×50 μL |
1 |
2 |
2 |
2.5 μL |
3 |
|
6-well |
2 mL |
2×125 μL |
2.5 |
5 |
5 |
5 μL |
7.5 |
|
60 mm |
5 mL |
2×250 μL |
5-10 |
10-20 |
10-20 |
12.5 μL |
20 |
|
10 cm |
10 mL |
2×500 μL |
15-25 |
30-50 |
30-50 |
25 μL |
40 |
|
T25 |
6 mL |
2×250 μL |
6-12 |
12-24 |
12-24 |
15 μL |
24 |
|
T75 |
15 mL |
2×750 μL |
20-40 |
40-80 |
40-80 |
37.5 μL |
60 |
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