Collagen from rat tail, Type I is prepared under aseptic conditions using the Birkedal-Hansen method, with a purity of ≥95%. It is dissolved in 0.006 mol/L acetic acid at a concentration of 4–5 mg/mL. This product can be used for coating cell culture vessels, especially for culturing cells that are difficult to adhere on ordinary cell cultureware. It can also be used to prepare three-dimensional collagen gels, allowing cells to grow in a simulated three-dimensional environment.
Quality Standards
1.Purity >95% analyzed by SDS-PAGE.
2.Sterility test result is negative.
3.After coating cell culture dishes at 2 μg/cm<sup>2</sup>, PC-12 cells show normal adhesion and growth.
4.At a concentration of 1 mg/mL and pH 7.0, this product forms 3D collagen gels with adequate strength. NIH-3T3 cells grow normally inside the 3D gels, and PC-12 cells grow normally on the surface of the 3D gels.
Directions for Use
1. Surface Coating of Cell Culture Vessels
Take a coating concentration of 2 μg/cm <sup>2</sup> as an example. Dilute the collagen to 0.012 mg/mL with sterile 0.006 mol/L acetic acid. Add the volume listed in the table below to the corresponding culture vessel, ensuring the collagen solution covers the entire surface. Remove the cap and air-dry overnight in a laminar flow hood. Alternatively, leave at room temperature for 1 hour, wash 3–4 times with PBS, and use immediately. Coated vessels can be stored at 4–25 °C for at least 3 months.
Culture Vessel | Surface Area (cm <sup>2</sup> , per well/dish) | Volume of 0.012 mg/mL Collagen (μL) | 96-well plate | 0.3 | 50 | 24-well plate | 1.9 | 300 | 12-well plate | 3.8 | 600 | 6-well plate | 9.5 | 1580 | 35 mm dish | 8 | 1330 | 60 mm dish | 21 | 3500 | 100 mm dish | 55 | 9170 |
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2. Preparation of 3D Collagen Gels
A. Preparation of Cell-Free 3D Collagen Gels
(Example: 1 mL of 1 mg/mL 3D gel)Add 200 μL of this product to a centrifuge tube in an ice bath. Add 690 μL of double-distilled water, then add to 12 μL of 0.1 mol/L NaOH.Note: Adding 12 μL of 0.1 mol/L NaOH into the collagen solution may cause local collagen gelation due to uneven mixing.Mix immediately. Add 100 μL of 10×PBS or 10×culture medium, mix well, and add to the culture vessel immediately.The pH will be approximately 7 after mixing. If phenol red is not included in PBS or medium, measure pH upon first use.Leave at room temperature for 20 min until gelation is complete, then transfer to an incubator. If 10×PBS is used, pre-equilibrate with an appropriate volume of cell culture medium before use.
B. Preparation of Cell-Containing 3D Collagen Gels
(Example: 1 mL of 1 mg/mL 3D gel)Prepare cells suspended in culture medium and keep in an ice bath. Add 200 μL of this product to 12 μL of 0.1 mol/L NaOH.Note: Adding 12 μL of 0.1 mol/L NaOH into the collagen solution may cause local collagen gelation due to uneven mixing.Mix immediately. Add 23 μL of 10×PBS or 10×culture medium and mix well.The pH will be approximately 7 after mixing. If phenol red is not included in PBS or medium, measure pH upon first use.Add 760 μL of cell suspension, mix gently, and add to the culture vessel immediately. Leave at room temperature for 20 min until gelation is complete. Add an appropriate volume of cell culture medium and transfer to an incubator.This product gels rapidly at neutral pH and room temperature. Keep samples cold during all procedures.
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