Reagents List
|
Product Name |
Cat.NO. |
|
40801ES |
|
|
Hieff™ Optimal-MEM Reduced Serum Medium |
41405ES |
|
DMEM High Glucose Medium（With L-glutamine, sodium pyruvate 110 mg/L. Free of HEPES, double-antibody) |
41401ES |
BMDMs Transfection Protocol
I. Cell Isolation and Culture
1. Preparation
- Mouse preparation: Euthanize mouse by cervical dislocation and immerse in 75% ethanol for approximately 10 min.
- Materials: Several sterile 10 cm culture dishes (for PBS tissue soaking), multi-well plates (for BMDM culture and transfection), DMEM culture medium, sterile PBS, red blood cell (RBC) lysis buffer; 2.5 mm injection needles and 20 mL syringes (for flushing bone marrow cells); centrifuge tubes; scissors and forceps.
2. Cell Isolation
1)Add PBS to a culture dish to cover the mouse legs.
2)Cut off the entire leg at the hip joint, remove the footpads, and carefully preserve the two main leg bones to prevent marrow leakage and cell contamination. Place the prepared legs into the PBS-filled dish. Use one dish per mouse to avoid cross-contamination.
3)Scrape off all muscle tissue from the bones, separate the joints, and place each leg (cut into two segments) into a fresh PBS-filled dish.
4)Soak the cleaned bones in 75% ethanol for 2 min for disinfection.
5)Rinse the bones thoroughly with PBS to remove residual ethanol.
6)Cut open the joint ends and flush the bone marrow from each bone into a centrifuge tube using a syringe filled with 5 mL PBS per bone.
7)Centrifuge at 1,500 rpm for 8 min, then discard the supernatant.
8)Add RBC lysis buffer, lyse red blood cells, then centrifuge again at 1,500 rpm for 8 min and discard the supernatant.
9)Resuspend the cell pellet in pre-warmed DMEM medium (volume adjusted based on the culture plate used) supplemented with M-CSF (final concentration: 25 ng/mL). Transfer the cell suspension directly into culture plates for differentiation into macrophages.
10)Gently shake the plate to ensure even cell distribution.
3. Cell Culture
Designate the day of isolation as Day 0. Change the medium on Day 3, Day 5, and Day 7 post-isolation. During medium changes, avoid vigorous pipetting; aspirate gently along the well wall.
Medium Change on Day 3:
1)Aspirate all old medium and replace with half the well's total volume of fresh DMEM.
2)Centrifuge the removed medium at 1,500 rpm for 8 min. Return half of the collected supernatant back to the well.
3)Add fresh M-CSF (final concentration: 25 ng/mL per well).
Medium Change on Day 5 and Day 7:
1)Aspirate all old medium and replace with half the well's total volume of fresh DMEM.
2)Centrifuge the removed medium at 1,500 rpm for 8 min. Discard half of the supernatant, resuspend the remaining pellet, and return it to the well.
3)Add fresh M-CSF (final concentration: 25 ng/mL per well).
II. Cell Transfection Procedure
Perform transfection directly in the culture plate on Day 7. Follow the protocol below:
1. DNA Transfection
[Note]: The optimal amount of transfection reagent may vary depending on cell type and experimental conditions. We recommend testing a range of reagent concentrations to determine the optimal dosage when using for the first time.
1)Plate cells so that they reach 70–90% confluency at the time of transfection.
2)Dilute LipoBooster 3000 transfection reagent in Opti-MEM medium according to the table below, and mix gently.
3)Dilute DNA in Opti-MEM medium to prepare the DNA pre-mix, then add Enhancer transfection enhancer solution, and mix gently to obtain diluted DNA.
4)Add the diluted DNA solution to the diluted LipoBooster 3000 solution (1:1 ratio).
5)Incubate the mixture at room temperature for 10–15 minutes.
6)Add the DNA–transfection reagent complexes dropwise to the cells, and gently mix.
2. siRNA Transfection
The siRNA transfection procedure follows the same steps as DNA transfection, except that the Enhancer transfection enhancer is not added when diluting siRNA.
3. mRNA Transfection
The mRNA transfection procedure and reagent volumes are the same as for DNA transfection, except that the Enhancer transfection enhancer is not added when diluting mRNA.
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 |
[Note]: The volumes provided in this table are for reference only and are suitable for both suspension and adherent cells. For suspension cells, the test can be performed at a cell density of 0.5–1×10⁶ cells/mL. The actual amounts of DNA and Booster DNA/RNA transfection reagent should be optimized depending on cell type and other experimental conditions. It is recommended to maintain a ratio between 1:0.5 and 1:5 (DNA : Booster transfection reagent). The amount and experimental conditions for mRNA are the same as those for DNA. If the cells are particularly fragile and excessive cell death is observed, better results may be achieved by reducing the amount of enhancer by half.
III. Analysis of Transfection Efficiency
When using green fluorescent protein (GFP) as the reporter, assess transfection efficiency by fluorescence microscopy and flow cytometry. When using luciferase (Luc) as the reporter, measure expression levels using a luciferase substrate assay kit.
1. Fluorescence Microscopy
Observe GFP expression by placing the culture plate under a fluorescence microscope.
2. Flow Cytometry Analysis
1)Detach cells from the well using a cell scraper.
2)Transfer the cell suspension to a centrifuge tube, centrifuge at 1,500 rpm for 8 min, and discard the supernatant.
3)Resuspend the cell pellet in 1 mL PBS, transfer to a 1.5 mL microcentrifuge tube, centrifuge at 1,500 rpm for 8 min, and discard the supernatant.
4)Resuspend cells in 100 μL PBS, add flow cytometry antibody, vortex gently, and incubate at 4°C for 10–30 min for macrophage surface staining.
5)After staining, add 1 mL PBS to each tube, centrifuge at 1,500 rpm for 8 min, and discard the supernatant.
6)Repeat the wash: add 1 mL PBS, centrifuge at 1,500 rpm for 8 min, and discard the supernatant.
7)Resuspend each pellet in 200–300 μL PBS, vortex gently, and analyze by flow cytometry.
3. Luciferase Assay Using Substrate Kit
1)Remove the existing culture medium from the wells.
2)Add an appropriate volume of lysis buffer to each well to lyse the cells.
3)Follow the instructions of the corresponding luciferase assay kit to measure luciferase expression levels.
IV. Experimental Results
Figure 1. Plasmid DNA transfection in primary bone marrow-derived macrophages (BMDMs) using Booster DNA&RNA Transfection Reagent versus L*3000. The result demonstrates superior primary cell transfection efficiency of Booster.
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