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User-generated protocol

Protocol: transfection of cerebellar slice cultures with Xfect reagent

Introduction Protocol Sample data

Introduction  

While primary culture of cells derived from the central nervous system is a powerful tool, the resulting lack of cellular heterogeneity or three-dimensional structure can render these approaches limited. One way to address these shortcomings is through the use of organotypic brain slice cultures, which preserve neural architecture and diversity for downstream analyses. Transfection of these cultures, however, can be both challenging and frustrating, which is why Dr. Maik Hintze (University of Bonn) developed this transfection protocol using the Xfect Transfection Reagent:

Cerebellar slice cultures are not very easy to transfect, often making viral transduction necessary. We have tested several transfection protocols that would give us good transfection efficiencies while allowing single-cell separation for counting and morphology. The Xfect Protocol provides a perfect balance of high cell numbers and good single-cell separation which perfectly matches our needs. The protocol is fast and simple to perform."

—Dr. Maik Hintze (University of Bonn)

Protocol  

A. Prior to transfection

  1. Prepare Culture Medium as follows:

    MEM, HEPES, no glutamine (Thermo Fisher Scientific, Cat. # 32360034) 100 ml
    L-Glutamine (200 mM) (Thermo Fisher Scientific, Cat. # 25030081) 1 ml
    HBSS, calcium, magnesium (Thermo Fisher Scientific, Cat. # 24020133) 50 ml
    Horse Serum, heat inactivated, New Zealand origin (Thermo Fisher Scientific, Cat. # 26050088) 50 ml
    Penicillin-streptomycin (10,000 units of each in 0.85% NaCl) 2 ml
    Total volume 203 ml
  2. Less than 4 hr prior to starting the transfection, transfer freshly prepared organotypic cerebellar slice cultures onto Millipore culture membranes (Biopore Membrane Filter roll, Hydrophilic PTFE; Millipore Sigma, Cat. # BGCM00010) and place on top of a Millipore culture dish insert (Millicell Cell Culture Insert, 30 mm, hydrophilic PTFE; Millipore Sigma, Cat. # PICMORG50). Allow slices to equilibrate in OptiMEM for at least 2 hours.

    NOTE: This dual-membrane approach improves transfection efficiency by slowing the rate at which the transfection reagent drains from the tissue into the underlying medium.

B. Prepare transfection mixture

Prior to beginning your transfection, consult the table below to determine the total required reagent volumes.

Required reagent volumes based on # of slices
5 10 15 20 25
DNA 4 µg 6 µg 10 µg 12 µg 16 µg
Xfect Reaction Buffer Bring to 23.8 µl Bring to 35.7 µl Bring to 59.5 µl Bring to 71.4 µl Bring to 95.2 µl
Xfect Polymer 1.2 µl 1.8 µl 3.0 µl 3.6 µl 4.8 µl
OptiMEM 75 µl 112.5 µl 187.5 µl 225 µl 300 µl
Total volume 100 µl 150 µl 250 µl 300 µl 400 µl
  1. Add DNA to a sterile 1.5-ml microcentrifuge tube and bring up to the indicated volume with Xfect Reaction Buffer. Mix by pipetting.
  2. Add the indicated volume of Xfect Polymer and mix well by vortexing for 5 sec.
  3. Incubate the mixture for 10 min at room temperature.
  4. During this incubation period, aliquot the indicated volume of OptiMEM (Thermo Fisher Scientific, Cat. # 31985062) into a separate, sterile, 1.5-ml microcentrifuge tube.
  5. After the 10-min incubation has passed, add the mixture from Step 3 to the OptiMEM and mix well by vortexing for 5 sec.

C. Transfect organotypic slice cultures

  1. Pipette 15 µl of the transfection mixture (Section B, Step 5) directly onto each prepared organotypic cerebellar slice (Section A, Step 2).
  2. While cerebellar slices are undergoing transfection, equilibrate Culture Medium for at least 2 hr.

    NOTE: As an optional step, you may wash each slice with 50–100 µl of OptiMEM 4 hr after transfection. Aspirate after 1 min. Use minimal OptiMEM in this step to avoid causing slices to float off the membrane/culture dish insert. Proceed immediately to Step 3.

  3. 4 hr after transfection, transfer membranes with organotypic slices from OptiMEM to a well containing the equilibrated Culture Medium.

Sample data  

Figure 1. Efficient transfection of organotypic cerebellar slice cultures with Xfect Transfection Reagent. Cerebellar slice cultures were generated from mouse pups (6–8 days old) and Xfect Transfection Reagent was used to transfect in an mRuby2-labeled calcineurin construct. Images shown were taken on Day 14 in vitro and are provided courtesy of Dr. Maik Hintze.


User-generated protocols

User-generated protocols

User-generated protocols are based on internal proof-of-concept experiments, customer collaborations, and published literature. In some cases, relevant results are discussed in our research news BioView blog articles. While we expect these protocols to be successful in your hands, they may not be fully reviewed or optimized. We encourage you to contact us or refer to the published literature for more information about these user-generated and -reported protocols. 

If you are looking for a product-specific, fully optimized User Manual or Protocol-At-A-Glance, please visit the product's product page, open the item's product details row in the price table, and click Documents. More detailed instructions for locating documents are available on our website FAQs page.

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Learn more about Xfect transfection

Overview of Xfect transfection

Xfect Transfection Reagent provides higher transfection efficiency, better cell viability, and a simple serum-compatible protocol.

Cell lines transfected with the Xfect reagent

See if your preferred cells have been successfully transfected with the Xfect reagent.

Transfection tips

Tips for maximizing transfection efficiency.

See all transfection reagent products

Select high-efficiency mammalian transfection reagents designed for plasmid DNA, RNA, or protein transfection.

Overview: Xfect RNA Transfection Reagent

Transfect all RNA types, including siRNA, mRNA, microRNA, and sgRNA, with Xfect RNA Transfection Reagent.

Xfect transfection protocol for cerebellar slices

Learn more about how to efficiently transfect cerebellar organotypic slice cultures.

Calcium phosphate transfection of neurons

Learn how to transfect your neuronal cultures more efficiently with lower toxicity.

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