CM cell collection
University of Pennsylvania
For Patients 18-19
Human cardiomyocyte dissociation.
Myocardial tissue was washed and resuspended in cold isolation buffer (130 mM NaCl; 5 mM KCl; 1.2 mM KH2PO4; 6 mM HEPES; 5 mM NaHCO3; 1 mM MgCl2; 5 mM Glucose) pH 7.4. Isolation buffer was supplemented with 0.36mM CaCl2 for enzyme activity. Cardiac tissue was incubated for 15 -20mins in isolation buffer supplemented with Collagenase IV (5mg/ml stock ,Sigma). After each incubation step, the supernatants were transferred to a tube and centrifuged at 600 rpm for 4 min. Cell pellets were resuspended in ice-cold isolation buffer. Several rounds of digestion were performed until tissue was fully digested.
Flow Cytometry and FACS.
Isolated cardiac cell suspensions obtained from human heart tissue were sorted on a FACSAria (Becton Dickenson Biosciences) instrument operating at low pressure (20 psi) using a 100µm nozzle. Initial size gates for forward scatter (FSC) vs. side scatter (SSC) were set to select the large cardiomyocytes with high FSC and SSC corresponding to larger and more granular cells. Stringent cell doublet discrimination was performed by a combination of high forward scatter height and area FSC-H/FSC-A and SSC-H vs. SSCW plots. Live single cells were selected by 7-aminoactinomycin D (7AAD, 1 µg/ml final concentration, Invitrogen) live/dead cell distinction staining. Finally, the live 7AAD –ve cells were distinguished. FACSDiVa Software was used for data acquisition and analysis.
First strand cDNA synthesis: (SCEmRNA-PT-18- SCEmRNA-HD-019)
The single cells were sorted individually into 96-well plates containing 5.25 µL of cold reverse transcription buffer [RT, dNTPs (2.5mM each), 5x first strand buffer, 100 mM DTT, a T7-oligo (dT) primer (10ng/µL), ERCC RNA Spike-In (1:4,000,000 dilution)]. The T7 portion of the T7-oligo (dT) primer incorporates the T7 RNA polymerase promoter in frame with the sequence antisense to the starting mRNA that is used later in the in vitro transcription (IVT) reaction. Cell lysis was performed by incubating at 70°C for 5 minutes in a PCR machine and immediately placing the plate on ice for at least 5 minutes. The volume of the reaction was adjusted to 10.25µL with nuclease-free water. The solution was then mixed and 1.75µL of RT enzyme mixture [Superscript III (200 U/µL) and Rnasin (40 U/µL)] was added followed by incubation at room temperature for 10 minutes. First strand cDNA synthesis was performed at 42°C for 30 mins in a PCR machine and the reaction was terminated by enzyme heat-inactivation at 70°C for 15 minutes. The molecular identity of the sorted cells was confirmed by PCR for positive expression of cardiomyocyte-specific gene (Tnnt2; troponin T2, cardiac) and negative expression of CD31 to exclude potential contamination by other cardiac cell types, such as cardiac fibroblasts and endothelial cells. The PCR confirmation involved two rounds of amplification (Round 1= 0.5µL of cDNA template and Round 2 = 10:90 dilution of cDNA from round 1) performed in 20µL reaction volumes using the Taq PCR core kit (Qiagen) according to the manufacturer's protocol. The 100bp 3´end PCR primers for Tnnt2 and CD31 were designed using Integrated DNA Technologies (IDT) PrimerQuest. Specific fragments were amplified with one cycle of 3 minutes at 94°C, 30 cycles of 30 seconds at 94°C, 30 seconds at 55°C, 1 minute at 72°C, and one cycle of 10 minutes at 72°C. PCR products (100bp long) were analyzed using 2% agarose ethidium bromide gel electrophoreses in TAE buffer at a constant voltage of 100 V for an hour and the amplicons were visualized with a ultraviolet transilluminator.
Single cell RT and First strand cDNA
synthesis RT mix:
|dNTPs(2.5mM each)||1.2 µl||61.2 µl||120 µl|
|5x 1st strand buffer||2.4 µl||122.4 µl||240 µl|
|T7-Oligo dT primer (10ng/µl)||0.3 µl||15.3 µl||30 µl|
|DTT (100mM)||0.45 µl||22.95 µl||45 µl|
|Nuclease free water||5.0 µl||255 µl||500 µl|
|ERCC RNA Spike In||0.9 µl||45.9 µl||90 µl|
|Total||10.25 µl||522.75 µl||1025 µl|
|Rnasin||0.3 µl||15.3 µl||30 µl|
|Superscript III||0.45 µl||22.95 µl||45 µl|
|Nuclease free water||1 µl||51 µl||100 µl|
|Total||1.75 µl||89.25 µl||175 µl|
- PCR machine with heated lid : 70ºC for 5 minutes
- Ice for 5 minutes
- Mix and spin
- Add 1.75 µl of Enzyme mix.
- PCR machine with heated lid : 42 ºC for 30 minutes, 70ºC for 15 minutes
- Store plate at -20 ºC or -80 ºC for longer periods.