Foetal cardiomyocyte size assessment

Material:

Animal-Free Blocker[R] and Diluent, R.T.U. (Vector laboratories Cat# SP-5035-100)

Anti-Cardiac Troponin T antibody [1C11] (Abcam Cat# ab8295)

Countess™ Cell Counting Chamber Slides (ThermoFisher Scientific Cat# C10312)

DAPI (Merck Cat# D9542)

Dulbecco′s Phosphate Buffered Saline (Merck Cat# D8537)

Falcon® 70 µm Cell Strainer (Corning Cat# 352350)

Foetal Bovine Serum (Gibco Cat# A5209)

Gibco Horse Serum, heat inactivated (ThermoFisher Scientific Cat# 26050088)

Goat Anti-Mouse IgG H&L (FITC) (Abcam Cat# ab6785)

Hepes (Merck Cat# 3375)

PhenoPlate 96-well (PerkinElmer Cat# 6055302)

Pierce™ Primary Cardiomyocyte Isolation Kit (ThermoFisher Scientific Cat# 88281)

Poly-L-Lysine Solution (0.01%) (Merck Cat# A-005-C)

Sterile 0.5- and 1.5-ml microcentrifuge tubes

Trypan blue stain 0.4% (Invitrogen Cat# T10282)

Wash Buffer 10x (Agilent Cat# S3006)

Wheat Germ Agglutinin, CF®640R Conjugate (Biotium Cat# BT29026-1)

Equipment:

Micro-centrifuge

Opera Phenix Plus High-Content Screening System 

Countess 3 Automated Cell Counter

1. Immerse excised foetal hearts (up to 2) in 500 µl HBSS (provided by the Pierce™ Primary Cardiomyocyte Isolation Kit manufacturer) in a 1.5-ml tube.
2. Mince hearts into small fragments and wash it twice with 500 μl ice-cold HBSS. Wait a minute for the tissue to precipitate between washes (leave tubes on ice).
3. Aspirate the HBSS and add 0.2 mL Cardiomyocyte Isolation Enzyme 1 (provided by the Pierce™ Primary Cardiomyocyte Isolation Kit manufacturer) and 10 μl Cardiomyocyte Isolation Enzyme 2 (provided by the Pierce™ Primary Cardiomyocyte Isolation Kit manufacturer)
4. Mix gently by flicking the tube and incubate in water bath at 37 °C for 30 minutes.
5. Remove the enzyme solution and wash tissue twice with 500 μl ice-cold HBSS. ATTENTION: the tissue is now digested and can be easily aspirated with the buffer. Proceed with caution.
6. Add 0.5 mL warm (37 ºC) dispersion Buffer [10% horse serum (heat inactivated), 5% FBS, 10 mM Hepes in HBSS (no Mg+ no Ca+)]. Pipette tissue up and down 25-30 times using a p1000 pipette.
7. Strain cells using a 70 µm strainer and transfer 450 µl of cell extract to a new 1.5 ml tube.
8. Add 450 µl 4% PFA solution (final: 2% PFA) and incubated for 18 min at room temperature (RT).
9. Centrifuge 2 min/700 g/RT.
10. Discard the fixative carefully through aspiration using a pipette.
11. Wash twice with 1 ml cold PBS, centrifuging 2 min/700 g/RT.
12. Resuspend cells in 200 ul PBS.
13. Mix 10 μl single-cell suspension with 10 μl 0.4% trypan blue in a new 0.5-mL microcentrifuge tube.
14. Transfer 12 μl trypan blue-stained cell suspension to a counting chamber.
15. Using a Countess system, count the total number of cells. Fixed cells can be stored in fridge for a few days.
16. Coat the bottom of the wells of a 96 well/plate with 50 μl 0.01% poly-L-lysine for 1 hour at room temperature.
17. Wash the wells 3 x with warm PBS.
18. Pipette ~ 5 x 10^4 cells into clean 0.5-ml tubes.
19. Centrifuge the tubes 2 min/700 g/RT and aspirate the buffer. From this point on, at the end of each incubation or wash, cells must be spun 2 min/700 g/RT, the supernatant aspirated and cells resuspended in the next solution by gentle flickering.
20. Incubate cells with 100 μl 5 μg/ml Wheat Germ Agglutinin in HBSS for 10 minutes
21. Wash with HBSS twice, incubating for 2 minutes.
22. Add Wash buffer and incubate for 10 min for cell permeabilization
23. Incubate with 100 μl animal-free blocking buffer for 45 min.
24. Remove blocking buffer and add 50 μl new animal-free blocking buffer solution containing primary antibody (Anti-Cardiac Troponin T antibody [1C11]) at 1:50 dilution and incubate for 1 hour.
25. Wash with wash buffer twice for 2 minutes.
26. Incubate with secondary antibody [Goat Anti-Mouse IgG H&L (FITC)] at 1:1000 dilution for 1 hour.
27. Wash with wash buffer twice for 2 minutes.
28. Incubate with DAPI solution
29. Aspirated DAPI solution and wash with 1 ml deionized water
30. Aspirate and add 100 ul deionized water.
31. Plate cells in coated plate overnight and scan using a High-Content Microscopy System (e.g.: Opera Phenix) on the next day.