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How to Culture HEK293 Cells: A Beginner's Guide
HEK293 cells are one of the most widely used cell lines in biomedical research—but keeping them healthy and happy requires attention to detail! Here’s a practical guide to culturing HEK293s, including essential lab supplies.

1. Key Lab Supplies You’ll Need
Culture Vessels: Tissue culture (TC) -treated flasks/plates (e.g., T75 flasks, 6-well plates).

Growth Medium: High-glucose DMEM supplemented with 10% Fetal Bovine Serum (FBS) and 1% Penicillin-Streptomycin.

Dissociation Agent: 0.25% Trypsin-EDTA (for detaching cells during passaging).

Cryopreservation Tools: Cryovials + freezing container (e.g., Mr. Frosty™) + DMSO.

Other Essentials: PBS (for rinsing), serological pipettes, and a humidified incubator (37°C, 5% CO₂).

2. Routine Maintenance & Passaging
When to Passage: When cells reach 80–90% confluency (avoid overgrowth!).

Trypsinization Tip: Add trypsin for 1–3 minutes at 37°C. Stop digestion immediately with fresh medium once cells round up.

Splitting Ratio: A 1:3 to 1:6 split ratio every 2–4 days works well for most HEK293 sublines.

3. Monitoring Cell Health
Healthy Morphology: Cells should appear cobblestone-like and adhere firmly to the surface.

Warning Signs: Granularity, elongation, or detachment signal stress or contamination.

4. Freezing & Thawing Protocols
Freezing Medium: 90% FBS + 10% DMSO. Cool cells slowly using a freezing container at -80°C before long-term storage in liquid nitrogen.

Thawing: Rapidly warm cryovials in a 37°C water bath (Less than 1 minute), dilute with pre-warmed medium, and centrifuge to remove DMSO.

5. Pro Tips for Success
Avoid Over-Confluency: HEK293s grow quickly—schedule passaging in advance!

Check for Contamination: Regularly inspect media for cloudiness or pH changes.

Use Quality Reagents: Consistent FBS batches improve reproducibility.

HEK293s are robust but demand consistent care. With the right tools and techniques, you’ll master their culture in no time! 🧫

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