EasySep™小鼠TIL（CD45）正选试剂盒
How to Extract CSF-Like Fluid from Choroid Plexus Organoids
How to Extract CSF-Like Fluid from Choroid Plexus Organoids
The STEMdiff™ Choroid Plexus Differentiation Kit efficiently and reproducibly generates human pluripotent stem cell-derived choroid plexus organoids featuring cystic structures containing fluid resembling cerebrospinal fluid (CSF). This organoid model permits access to CSF-like fluid without requiring invasive lumbar puncture procedures, and maintains a blood-free sample. Extracting the fluid from these organoids presents a valuable tool for disease-related biomarker discovery and blood-CSF permeability assays. The following protocol describes two options for the extraction of CSF-like fluid from choroid plexus organoids, and was adapted from a previously published protocol developed by Pellegrini et al1.
Table 1. Comparison of CSF-like Fluid Extraction Methods
Protocol Option 1: Syringe Extraction
This method works best with larger choroid plexus organoids (> 1 mm in diameter). Expected extracted volumes are 100 - 1000 µL per organoid.
Materials
- Syringe (1 mL or 3 mL) e.g. (Covidien Catalog #8881501400 or #8881513934)
- Needle (28G or smaller) e.g. (BD Eclipse™ Catalog #305757)
- 1.7 mL microcentrifuge tubes (e.g. Catalog #38089)
- D-PBS (Without Ca++ and Mg++) (Catalog #37350)
- Pipettors (e.g. Corning® Lambda™ Plus Pipettor, Catalog #38058 and Catalog #38059)
- Pipette tips (e.g. Corning® Filtered Pipette Tips, Catalog #38031 and Catalog #38032)
Procedure
- Transfer a single ChP organoids using a cut P1000 pipette tip (or sterile spatula) to an appropriate vessel to wash (for small to medium ChP organoids these can be transferred to a 12-well plate, for large ChP organoids these can be transferred to a 6-well plate).
- Rinse ChP organoid with enough D-PBS to cover the organoid (0.5 to 3 mL depending on vessel and organoid size).
- Perform wash a minimum of three times to remove excess media, immediately removing wash each time. Note: perform wash steps gently to prevent popping of the ChP organoid cyst.
- Use a 200 μL or 1000 μL pipette to remove as much PBS as possible to prevent dilution of the CSF-like fluid.
- Use a syringe + needle assembly to poke the choroid plexus organoid and slowly extract CSF-like fluid. Aim for the middle of the cysts if possible. Holding the tube at eye-level can make this easier.
- Expel CSF-like fluid into a new microcentrifuge tube and store at -20°C to -80°C for subsequent analysis.
- Note: The total volume of CSF-like fluid extracted from each organoid can range from 10 - 1000 µL, depending on the method.
- The deflated choroid plexus organoid can be used as a control in subsequent experiments.
Protocol Option 2: Centrifugation Extraction
This method works best with smaller choroid plexus organoids (< 1 mm in diameter) mm or when multiple ChP organoids need to be processed per sample. It’s not recommended to use this method with organoid cyst diameter greater than 1 mm due to dimensions of the Amicon Ultra-0.5 mL Centrifugal Filter. Expected extracted volumes are 10 - 500 µL per organoid.
Materials
- Amicon® Ultra-0.5 Centrifugal Filter Unit 100 kDa cut-off (Amicon Catalog #UFC5100)
- 1.7 mL microcentrifuge tubes (e.g. Catalog #38089)
- D-PBS (Without Ca++ and Mg++) (Catalog #37350)
- Pipettors (e.g. Corning® Lambda™ Plus Pipettor, Catalog #38058 and Catalog #38059)
- Pipette tips (e.g. Corning® Filtered Pipette Tips, Catalog #38031 and Catalog #38032)
Procedure
- Transfer ChP organoids using a cut p1000 pipette tip (or sterile spatula) to an appropriate vessel to wash
- Note: Small to medium ChP organoids can be transferred to a 12-well plate, and large ChP organoids can be transferred to a 6-well plate.
- Rinse ChP organoid with enough D-PBS to cover the organoid (0.5 to 3 mL depending on vessel and organoid size).
- Perform wash a minimum of three times to remove excess media, immediately removing wash each time. Note: perform wash steps gently to prevent popping of the ChP organoid cyst.
- Using a cut wide-bore pipette tip, transfer choroid plexus organoid(s) to a Amicon Ultra-0.5 mL Centrifugal Filter. Remove as much excess PBS as possible to prevent dilution of the CSF-like fluid.
Note: 2 to 3 organoids may be loaded into one tube. More than this may clog the filter.
- Centrifuge the tube at 10,000g for 2 minutes.
- CSF-like fluid is collected as the flow-through and can be store at -20°C to -80°C for subsequent analysis. The deflated choroid plexus organoid can be used as a control in subsequent experiments.
Analysis of CSF-Like Fluid
CSF-like fluid may be analyzed by SDS-PAGE and Western blotting using the following antibodies:
- Rabbit anti-IGF-2 (Abcam ab204567) 1:250
- Goat Anti-Rabbit IgG H&L (HRP) (ab205718) (1:2000)
- HRP-Clusterin (Biolegend 848703) 1:250
CSF-like fluid may also be analyzed using other methods such as mass spectrometry, as demonstrated in Pellegrini et al1.
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