2D cell culture MBG 2020 - Manchester BIOGEL · 2020. 7. 2. · § Transfer 0.2 mL of the...

PROTOCOL 2D Cell Culture in PeptiGels ® This protocol describes the use of PeptiGels ® for 2-dimensional (2D) cell culture. We highly recommend the use of a positive displacement pipette (such as the Gilson piston pipette) to allow easy pipetting as these are viscous gels. We recommend the use of cell inserts (such as the Greiner Bio-One cell inserts or equivalent) to increase gel stability and media diffusion. As a guide, this protocol has been written for a total volume of 0.2 mL PeptiGel ® . Please scale up or down according to culture requirements. 2D Cell Culture Protocol § Pre-wet the inserts in media/PBS for 1 hour to prevent bubbles getting trapped into the membrane pores. § Remove PeptiGel ® from the fridge and pre-warm to room temperature. Hint: If required, centrifuge PeptiGel ® for 1 minute at 2500 g (3000 rpm) to remove air bubbles. § Pipette the PeptiGel ® in sufficient quantity to cover the surface of the insert. As a guide, 0.2 mL PeptiGel ® is enough for a 24 well plate insert. Hint: Gently tap the plate against a sterile surface approximately 20 times to obtain a flat PeptiGel ® surface. Hint: If necessary, centrifuge the plate containing the insert for 1 minute at 2500 g (3000 rpm) to obtain a flat surface. § Add 1 mL of culture media to the well containing the inserts and incubate at 37 o C for 30 minutes. § Add 0.2 mL of culture media to the surface of pre-conditioned PeptiGel ® in the insert and incubate at 37 o C for 30 mins. Hint: Pre-conditioning ensures the pH of PeptiGel ® becomes neutral (pH 7). Hint: Pre-conditioning also ensures a uniform distribution of media nutrients before cell seeding. § Resuspend your cells to the required cell density in 0.2 mL of culture media. § Remove the media used to pre-condition PeptiGel ® . Hint: Leave some media on the surface of pre-conditioned PeptiGel ® to prevent the pipette tip disrupting the PeptiGel ® .

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Transcript of 2D cell culture MBG 2020 - Manchester BIOGEL · 2020. 7. 2. · § Transfer 0.2 mL of the...

Page 1: 2D cell culture MBG 2020 - Manchester BIOGEL · 2020. 7. 2. · § Transfer 0.2 mL of the resuspended cell suspension on top of the PeptiGel® and wait for 5 minutes. § Add 1 mL

PROTOCOL

Alderley Park | Cheshire | SK10 4TG

2D Cell Culture in PeptiGels®

This protocol describes the use of PeptiGels® for 2-dimensional (2D) cell culture. We highly recommend the use of a positive displacement pipette (such as the Gilson piston pipette) to allow easy pipetting as these are viscous gels. We recommend the use of cell inserts (such as the Greiner Bio-One cell inserts or equivalent) to increase gel stability and media diffusion. As a guide, this protocol has been written for a total volume of 0.2 mL PeptiGel®. Please scale up or down according to culture requirements.

2D Cell Culture Protocol

§ Pre-wet the inserts in media/PBS for 1 hour to prevent bubbles getting trapped into the membrane pores.

§ Remove PeptiGel® from the fridge and pre-warm to room temperature. Hint: If required, centrifuge PeptiGel® for 1 minute at 2500 g (3000 rpm) to remove air bubbles.

§ Pipette the PeptiGel® in sufficient quantity to cover the surface of the insert. As a guide,

0.2 mL PeptiGel® is enough for a 24 well plate insert. Hint: Gently tap the plate against a sterile surface approximately 20 times to obtain a flat PeptiGel® surface. Hint: If necessary, centrifuge the plate containing the insert for 1 minute at 2500 g (3000 rpm) to obtain a flat surface.

§ Add 1 mL of culture media to the well containing the inserts and incubate at 37oC for 30 minutes.

§ Add 0.2 mL of culture media to the surface of pre-conditioned PeptiGel® in the insert and incubate at 37oC for 30 mins.

Hint: Pre-conditioning ensures the pH of PeptiGel® becomes neutral (pH 7). Hint: Pre-conditioning also ensures a uniform distribution of media nutrients before cell seeding.

§ Resuspend your cells to the required cell density in 0.2 mL of culture media.

§ Remove the media used to pre-condition PeptiGel®.

Hint: Leave some media on the surface of pre-conditioned PeptiGel® to prevent the pipette tip disrupting the PeptiGel®.

Page 2: 2D cell culture MBG 2020 - Manchester BIOGEL · 2020. 7. 2. · § Transfer 0.2 mL of the resuspended cell suspension on top of the PeptiGel® and wait for 5 minutes. § Add 1 mL

§ Transfer 0.2 mL of the resuspended cell suspension on top of the PeptiGel® and wait for 5 minutes.

§ Add 1 mL of fresh culture media to the plate and incubate overnight.

§ Next day, change the media and repeat as necessary depending on your cell requirements.

Support For further support, please contact our technical support team on +44 (0) 1625 238 800 or [email protected]. Please note there are also useful supporting videos on our website (www.manchesterbiogel.com/resources/technicalvideos). Disclaimer All standard safety procedures regarding cell culture need to be observed.

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