Cell Transfection | Experimental Protocol for Primary Extraction, Cult

I. Cell Extraction and Culture

1. Preparation:

1) Mouse Handling: Sacrifice mice by cervical dislocation, then immerse in 75% ethanol for approximately 10 min.

2) Experimental Supplies: Several clean 10 cm culture dishes for tissue soaking in PBS during the experiment; multi-well plates for culturing BMDMs and transfection; DMEM medium, sterile PBS, Red Blood Cell Lysis Buffer; 2.5 mm needle and 20 mL syringe for flushing cells from mouse bone marrow; centrifuge tubes; scissors and forceps.

2. Cell Extraction:

1) Add enough PBS to a culture dish to submerge the mouse legs.

2) Cut off the entire leg from the mouse at the hip joint, remove the foot. Avoid crushing the two main leg bones to prevent marrow leakage and cell contamination. Place the trimmed leg into the culture dish for soaking. Note: Use one dish per mouse to prevent cross-contamination.

3) Remove all muscle tissue from the bones, separate the joints. Place the two bone segments per leg into a new dish with clean PBS.

4) Immerse the cleaned bones in 75% ethanol for 2 min for sterilization.

5) Rinse the bones with PBS to remove the ethanol.

6) Cut open the joints. Use the syringe to flush the bone marrow from the bones into a centrifuge tube using PBS (typically 5 mL PBS per bone).

7) Centrifuge at 1500 rpm for 8 min. Discard the supernatant.

8) Add Red Blood Cell Lysis Buffer. After lysis, centrifuge at 1500 rpm for 8 min. Discard the supernatant.

9) Add culture medium to resuspend the cell pellet (medium volume calculated based on the specific plate used). Add M-CSF (final concentration 25 ng/mL). Directly add the cell suspension to the multi-well plate to induce differentiation into macrophages.

10) Gently swirl the plate to ensure even cell distribution.

3. Cell Culture:
Designate the extraction day as Day 0. Change the medium on Day 3, Day 5, and Day 7 after BMDM extraction. Take care not to pipette vigorously during medium changes; gently aspirate along the wall of the dish!

Day 3 Medium Change Process:

1) Aspirate all the culture medium, replenish with half the plate volume of fresh DMEM.

2) Centrifuge the aspirated medium at 1500 rpm for 8 min, return half of the supernatant to the well.

3) Supplement with M-CSF (final concentration per well: 25 ng/mL).

Day 5 and Day 7 Medium Change Process:

1) Aspirate all the culture medium, replenish with half the plate volume of fresh DMEM.

2) Centrifuge the aspirated medium at 1500 rpm for 8 min, discard half of the supernatant, resuspend the cell pellet in the remaining volume, and return it to the well.

3) Supplement with M-CSF (final concentration per well: 25 ng/mL).

II. Cell Transfection Experiment

Perform the transfection experiment directly in the culture plate on Day 7. Follow the experimental method below.

1. DNA Transfection
Note: The amount of transfection reagent used is influenced by cell type and other experimental conditions. It is recommended to perform a gradient optimization for the optimal amount during initial use.

1) Cell Seeding: At the time of transfection, a cell confluence of 70%-90% is ideal.

2) Dilute the Booster DNA/RNA Transfection Reagent solution in Opti-MEM Reduced-Serum Medium according to the table below. Mix gently.

3) Dilute the DNA in Opti-MEM Medium to obtain a DNA premix. Then add the Transfection Enhancer solution. Mix gently to obtain diluted DNA.

4) Add the diluted DNA to the diluted Booster DNA/RNA Transfection Reagent solution (1:1 ratio).

5) Incubate at room temperature for 10-15 minutes.

6) Add the DNA-transfection reagent complexes dropwise to the cells. Gently swirl the plate to mix.

2. siRNA Transfection
The procedure for siRNA transfection is the same as for DNA transfection. However, do not add the Transfection Enhancer when diluting the siRNA.

3. mRNA Transfection
The procedure and amounts for mRNA transfection are the same as for DNA transfection. However, do not add the Transfection Enhancer when diluting the mRNA.

4. Transfection Amounts for Different Culture Vessels (For Reference Only):

Culture Vessel	Medium Volume	Opti-MEM Volume in Complex	DNA (μg)	Booster Transfection Reagent(μL)	Enhancer Transfection Enhancer (μL)	siRNA Volume (Initial Conc. 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 amounts in this table are for reference only. The specific amounts of DNA and Hieff Trans™ Booster need to be optimized based on cell type and other experimental conditions.

It is recommended to maintain the ratio between 1:0.5 and 1:5. The usage and experimental conditions for mRNA are the same as for DNA. If cells are fragile leading to significant cell death, better results may be obtained by halving the amount of Enhancer.

III. Cell Transfection Result Testing

When using Green Fluorescent Protein (GFP) as the test protein, use a fluorescence microscope and flow cytometry for testing.

When using Luciferase (Luc) as the test protein, use a luciferase substrate kit for testing.

1. Fluorescence Microscope Observation
Place the plate under a fluorescence microscope to observe GFP expression.

2. Flow Cytometry Analysis

1) Detach cells in the well using a cell scraper.

2) Aspirate the cell suspension into a centrifuge tube. Centrifuge at 1500 rpm for 8 min. Discard the supernatant.

3) Resuspend the cell pellet in 1 mL PBS per tube. Transfer the cell suspension to a 1.5 mL centrifuge tube. Centrifuge at 1500 rpm for 8 min. Discard the supernatant.

4) Resuspend cells in 100 μL PBS. Add flow cytometry antibodies, vortex, and incubate at 4°C for 10-30 min to stain macrophages.

5) After staining, add 1 mL PBS to each tube, centrifuge at 1500 rpm for 8 min. Discard the supernatant.

6) Add 1 mL PBS to each tube, centrifuge at 1500 rpm for 8 min. Discard the supernatant.

7) Resuspend cells in a certain volume of PBS (200-300 μL) per tube, vortex, and proceed with flow cytometry analysis.

3. Luciferase Substrate Kit Testing

1) Remove the original medium from the wells.

2) Add tissue/cell lysis buffer to each well to lyse the cells.

3) Test luciferase expression levels according to the corresponding kit instructions.

IV. Experimental Results

Figure. Plasmid DNA transfection in primary bone marrow-derived macrophages (BMDMs) using Booster DNA&RNA Transfection Reagent versus competitor L*3000. The result demonstrates superior primary cell transfection efficiency of Booster.