Protocol for Transfection of Primary Bone Marrow-Derived Macrophages (BMDMs)

1. Experimental Materials

  • BMDM Cells
  • Target plasmid DNA (Concentration ≥ 1000 ng/μL)
  • Hieff TransTM Booster DNA&RNA Transfection Reagent (Cat#40801)（Know more and get Free Sample）
  • Opti-MEM Reduced Serum Medium
  • Compatible tissue culture plates and related lab consumables (using a 24-well plate as an example)

2. Experimental Procedure

1) Preparation One Day Before Transfection

Count cells and seed according to their growth rate to ensure cell density reaches approximately 80%~90% at the time of transfection the next day.

2) Pre-treatment on Transfection Day (Optional)

Aspirate the cell culture medium and replace it with 0.5 mL of fresh complete medium.

3) Preparation of DNA-Transfection Reagent Complex

  • Dilute DNA: For each well, take 0.5 μg DNA and add it to 25 μL Opti-MEM Reduced Serum Medium and 1 μL Transfection Enhancer.
  • Dilute Transfection Reagent: Take 1 μL Transfection Reagent and add it to 25 μL Opti-MEM Reduced Serum Medium.
  • Form DNA-Transfection Reagent Complex: Mix the diluted DNA (26 μL) and the diluted Transfection Reagent (26 μL). Incubate at room temperature for 10-15 minutes.

4) Cell Transfection

Add the DNA-transfection reagent complex dropwise to the cells. Gently rock the dish back and forth to ensure even distribution.

5) Post-Transfection Handling

Do not remove the complex. Continue culturing in a 37°C CO₂ incubator for 18-24 hours. Proceed to detect target gene expression afterwards.

3. Experimental Result

Figure 1. 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.

 Figure 1. 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.

4. Customer Feedback

1) High-Efficiency Primary Dermal Fibroblasts Transfection

 Figure 6. Plasmid EGFR DNA transfection in primary dermal fibroblasts using Booster DNA&RNA Transfection Reagent versus L*3000. The result demonstrates superior primary cell transfection efficiency of Booster.

 Figure 2. Plasmid EGFR DNA transfection in primary dermal fibroblasts using Booster DNA&RNA Transfection Reagent versus L*3000. The result demonstrates superior primary cell transfection efficiency of Booster.

2) High-Efficiency Porcine Alveolar Macrophages Transfection

Figure 6. DNA transfection in primary Porcine Alveolar Macrophages (PAMs) using Booster DNA&RNA Transfection Reagent versus competitor L*3000. The result demonstrates superior primary cell transfection efficiency of Booster.

Figure 3. DNA transfection in primary Porcine Alveolar Macrophages (PAMs) using Booster DNA&RNA Transfection Reagent versus L*3000. The result demonstrates superior primary cell transfection efficiency of Booster.

3) High-Efficiency Primary Mouse Hepatocytes Transfection

Figure 4. Plasmid DNA transfection in Primary mouse hepatocytes using Booster DNA&RNA Transfection Reagent versus T brand L*3000. The result demonstrates superior primary cell transfection efficiency of Booster.

4) High-Efficiency Primary mouse neural stem cells Transfection

Figure 3. mRNA transfection in Primary mouse neural stem cells using Booster DNA&RNA Transfection Reagent versus S brand S*8008. The result demonstrates superior primary cell transfection efficiency of Booster.

Figure 5. mRNA transfection in Primary mouse neural stem cells using Booster DNA&RNA Transfection Reagent versus S brand S*8008. The result demonstrates superior primary cell transfection efficiency of Booster.

5. Frequently Asked Questions (FAQs) & Solutions

1) How to improve transfection efficiency?

  • Optimize nucleic acid vector structure (promoter selection, vector backbone optimization).
  • Choose a transfection reagent validated for primary cells.

Hieff TransTM Booster DNA&RNA Transfection Reagent(Cat#40801) has the following features and supports free samples.

  • Low Cytotoxicity: The novel polymer replaces traditional liposomes to reduce cytotoxicity and improve cell viability.
  • Fast Release and Higher Efficiency: Fast release minimizes intracellular nucleic acid degradation, achieving >90% transfection efficiency (measured by flow cytometry).
  • Premium Options for Difficult-to-transfect cell lines: Sensitive and primary cells.
  • Proven Performance, Broad Cell Line Coverage: Achieves high efficiency in 200+ various cell lines including 293T, HeLa, MCF7, HepG2, A549, NIH3T3, RAW264.7, and HCT116 and primary cells, with consistent and reproducible results.
  • One Reagent, All Nucleic Acids: Delivers DNA, siRNA, miRNA, mRNA, and ASO with high efficiency—even up to 15 kb fragments.

2) What to do if transfection results are abnormal?

Set up internal reference control groups (e.g., use a constitutively expressing plasmid to rule out systematic errors).

Detect cell viability (confirm mortality <20% using Calcein-AM/PI double staining).

Related Product

Application

Name

Catalog No.

Size

Works with DNA, siRNA, miRNA, mRNA, and ASO, etc.

Proven success in primary cells.

Hieff TransTM Booster DNA&RNA Transfection Reagent

40801

100 μL/1.5 mL

 

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