Notice: This protocol is not supported by Parse Biosciences. This Customer Developed Protocol is provided for general information only and is not directly supported, endorsed, or certified by Parse Biosciences. Parse Biosciences gives no warranties and makes no claims about the provided protocol.
Version 1.0
A PDF version of this protocol is available for download here.
Single-cell isolation from embryonic chicken heart
Developed by Iwijn De Vlaminck and Jonathan T. Butcher
About the De Vlaminck and Butcher labs
The Iwijn De Vlaminck lab uses spatiotemporal single cell RNA sequencing to investigate questions related to health and development. The lab specializes in work on infectious and immune-mediated diseases, as well as spatial profiling of microbiomes and infection in complex tissues.
The Jonathan T. Butcher lab studies how mechanical forces shape the developing heart and understanding how to apply these principles to human health. Their main focuses within development are on cardiovascular developmental biomechanics, and within health they study reactivation of developmental biology in valve diseases as well as tissue engineering and regeneration.
This protocol was used in
Spatiotemporal Atlas of Heart Development Reveals Blood-Flow-Dependent Cellular, Structural, Metabolic, and Spatial Remodeling. Jooyoung Park, Shuofei Sun, Rohit Agarwal, Andreas Stephanou, Mong Lung Steven Poon, Hyun Maeng, Peyton Lancaster, Iwijn De Vlaminck, Jonathan Butcher
bioRxiv 2025.12.09.693024; doi: https://doi.org/10.64898/2025.12.09.693024
Materials
- Material | Supplier | Part Number | Notes
- Centrifuge with swinging bucket rotor | Varies | Varies | Compatible with 15 mL or 5 mL centrifuge tubes and capable of reaching 4°C.
- Shaker | Varies | Varies | Compatible with 1.5mL tubes
- 1.5 mL Protein LoBind® Tubes | Eppendorf® | 022431081 | Or equivalent
- 40 μm cell strainer | Corning® | 431750 | Or equivalent
- Collagenase, Type II, powder | Thermo Fisher Scientific® | 17101015
- Hanks′ Balanced Salt solution | Merck® | H9394 | Or equivalent
- RNaseZAP® RNase Decontamination Solution | Thermo Fisher Scientific | AM9780
Preparation
1. Clean the bench top and dissection area with 70% ethanol, followed by RNaseZAP to remove RNases.
2. Prepare the following solutions, and add reagents in the order provided in the table.
Reconstituted collagenase II stock example
Note: Each lot will have a different value of U/mg, so make note of the final concentration when reconstituted in 1mL
- Reagents | Stock Concentration | Final Concentration | Total amounts
- Collagenase II powder | 265 U/mg | 265 U/µL | 1 g
- Hank’s Balanced Salt Solution | N/A | N/A | 1 mL
Working concentration collagenase II example
Note: Each lot will have a different value of U/mg, so adjust according to the true concentration of your stock.
- Reagents | Stock Concentration | Final Concentration | Total amount for 1 mL
- Reconstituted collagenase II stock | 265 U/µL | 300 U/mL | 1.13 µL
- Hank’s Balanced Salt Solution | N/A | N/A | 1 mL
Procedure
1. Dissect hearts and place in ice-cold Hank’s Balanced Salt Solution
2. For hearts from embryos 7 days old or less, place the hearts directly in 500 μL of 300 U/mL collagenase type II in a 1.5 mL tube and incubate for 15 minutes at 37°C on a shaker. For hearts of embryos older than 7 days, chop hearts into small pieces using a razor blade and then transfer into 1 mL of 300 U/mL collagenase type II in a 1.5mL tube and incubate for 20 minutes at 37°C on a shaker.
3. Centrifuge and remove supernatant
Note: Centrifugation conditions may vary between instruments. Users should ensure that all centrifugation steps result in cells suitable for analysis before proceeding with fixation and barcoding.
4. For embryos 7 days old or younger, resuspend cells in 500 μL of collagenase II and incubate for 10 minutes at 37°C on a shaker. For embryos older than 7 days, resuspend cells in 500 μL of collagenase II and incubate for 10 minutes at 37°C on a shaker.
5. Pass cells througha 40 µm strainer into a new 1.5 mL tube
6. Centrifuge and resuspend in PBS
Note: Red blood cell depletion may be required. It is recommended to assess the fraction of red blood cells before proceeding.
7. Count cells and assess quality
Note: Accurate counting and assessment of cells is critical to generate high-quality data. If using a hemocytometer, use all four quadrants for accuracy. View at 10, 20, and 40X for morphology and debris. Optimally, less than 5% aggregation and 5% debris. A best practices resource for hemocytometer use, see: https://www.hemocytometer.org/
8. Proceed to the appropriate Parse Biosciences Cell Fixation Kit User Guide (either standard Cell Fixation, Low-Input Fixation).
Notice
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