Norepinephrine [(±)-arterenol], mw 319.3
- Norepinephrine is made up in 30 mM ascorbic acid.
- Make up 100 mL of 30 mM ascorbic acid by adding 0.59 g ascorbic acid to 100 mL of cell culture grade distilled water.
- Add 80 mg norepinephrine to 25 mL of the 30 mM ascorbic acid.
- Filter-sterilize using a 0.2 µm Acrodisc syringe filter.
- Aliquot in 1 mL volumes into sterile microtubes with screw caps, and store at -20 °C. This is 10 mM (stock) norepinephrine. Use 1 mL of stock per 100 mL medium for a 0.1 mM final concentration.
- Norepinephrine needs to be made up fresh monthly.
- L-Glutamine comes as a 100x solution, and is aliquoted into working volumes and frozen.
- Freezing medium is made up of 95% FBS/5% DMSO.
- This can be stored up to a week at 4 °C.
Soybean Trypsin Inhibitor
- Weigh out 25 mg of soybean trypsin inhibitor, and place into a beaker containing 100 mL of Dulbecco’s phosphate buffered saline (PBS; Ca2+-free and Mg2+-free) until dissolved.
- Filter-sterilize, using a 0.2 µm syringe filter, into a 100 mL bottle.
- This is good for a month at 4 °C.
- Weigh out 0.1 g gelatin and place into a 500 mL glass bottle.
- Add distilled water to the 500 mL mark, and autoclave. This gelatin will go into solution while being autoclaved. The concentration of the gelatin is 0.02%.
- Fibronectin (1 mg/mL) is received in a tube as a liquid. Dilute 1 mL fibronectin in 199 mL of 0.02% gelatin. Mix gently, and immediately aliquot 6 mL into each labeled 15 mL centrifuge tube. Freeze aliquots at -20 °C.
- Before culturing cells, coat tissue culture flasks with gelatin/ fibronectin (1 mL/T25 or 3 mL/T75 flask). Cap the flasks, and incubate at 37 °C for at least an hour.
- Remove the gelatin/fibronectin by aspiration just before adding cells to the flasks.
- Cultures are fed (5 mL/T25 flask) with supplemented Claycomb Medium every weekday.
- To avoid feeding the cells on weekends, 10 mL of supplemented Claycomb Medium is added to each T25 flask on Friday afternoons; this medium is not changed until the following Monday morning.
Passaging – Procedure for a 1:2 Split
- After the cells first arrive, it is recommended that they be split when they reach confluency.
- Split each of the T25 flasks 1:2, resulting in four T25 flasks. One of these four T25 flasks will be your “working” set of cells.
- When the cells in the remaining three T25 flasks reach confluency three days later, they will be split 1:3, resulting in 3 T75 flasks to be frozen. We recommend freezing away at least 3 T75 flasks.
- Thus, within a week of receiving the HL-1 cells, you should have three cryovials of these cells frozen away for future use.
- It is recommended that cultures be split only after reaching full confluency.
- Rinse each T25 flask briefly with 3 mL of phosphate buffered saline (PBS) warmed to 37 °C (use 6 mL for T75) by pipetting the PBS onto the base of the flask (side opposite the cap), trying not to hit cells directly. Rinse gently and remove by aspiration.
- Add 1 mL of pre-warmed 0.05% trypsin/EDTA per T25 flask (3 mL per T75). Incubate at room temperature for 1 minute.
- Remove and add fresh 0.05% trypsin/EDTA. Incubate at room temperature for an additional 2 minutes.
- Examine microscopically and, if cells are still adhered, rap the flask on the benchtop to dislodge remaining cells.
- To inactivate the enzyme, add an equal amount (1 mL per T25) of soybean trypsin inhibitor.
- Transfer cells from the flask into a 15 mL centrifuge tube.
- Rinse the empty flask with 5 mL wash medium (Claycomb Medium containing only 5% FBS and penicillin/ streptomycin), and add to the cells already in the 15 mL centrifuge tube.
- Centrifuge at 500×g for 5 minutes.
- Meanwhile, remove the gelatin/fibronectin solution from each T25 flask, and add 4 mL supplemented Claycomb Medium/ flask. Set aside.
- Remove the tube containing the HL-cardiomyocytes from the centrifuge. Remove the supernatant by aspiration, and gently resuspend the pellet in 2 mL of supplemented Claycomb Medium.
- Transfer 1 mL of the cell suspension into each of two labeled, gelatin/fibronectin-coated T25 flask. Each flask now contains 5 mL.
- If the cells are passaged on a Friday, use 2x the volume of supplemented Claycomb Medium per flask.
IT IS RECOMMENDED THAT YOU FREEZE 3 OR MORE VIALS AS SOON AS POSSIBLE AFTER RECEIPT OF THE CELLS (please see note under “Passaging”). This allows you to return to this passage, and also protects you in case of contamination.
- We generally freeze the contents of one confluent T75 flask into one cryovial. (When cells are needed, this cryovial is thawed into one T75 flask.)
- Briefly rinse the T75 flask containing the HL-1 culture with 5 mL of PBS warmed to 37 °C. Remove by aspiration.
- Transfer 3 mL of 0.05% trypsin/EDTA into the flask.
- Incubate the flask at 37 °C for 1 minute.
- Remove the trypsin/EDTA from the flask, and replace with 3 mL of fresh 0.05% trypsin/EDTA.
- Incubate at 37 °C for 2 minutes.
- Check under a microscope that cells are dislodged. If not, rap the flask on the benchtop to dislodge any adherent cells.
- Add 3 mL of soybean trypsin inhibitor to the flask, and transfer the 6 mL into a 15 mL centrifuge tube.
- Rinse each empty flask with 8 mL wash medium, and add to the cells already in the 15 mL centrifuge tube. Total volume is now 14 mL.
- Centrifuge tube for 5 minutes at 500×g.
- Remove wash medium by aspiration.
- Gently resuspend each pellet in 1.5 mL of freezing medium (95% FBS/5% DMSO).
- Pipette resuspended cells into a cryovial. Place the cryovial containing the cells into a Nalgene freezing jar containing room temperature isopropanol.
- Immediately place the freezing jar into a –80 °C freezer, which allows the cells to freeze at a rate of -1 °C/minute.
- Six to twelve hours later, transfer the vial to a liquid nitrogen dewar.
- Gelatin/fibronectin-coat a T75 tissue culture flask for at least an hour in a 37 °C incubator.
- Remove the gelatin/fibronectin from the culture flask, and replace with 10 mL of supplemented Claycomb Medium. Place this flask back into incubator.
- Transfer 10 mL wash medium into an empty 15 mL centrifuge tube. Incubate tube in a 37 °C water bath.
- Quickly thaw the cells in a 37 °C water bath (about 2 min), and transfer into the 15 mL centrifuge tube containing the wash medium.
- Centrifuge for 5 minutes at 500×g.
- Remove the tube from the centrifuge and remove the wash medium by aspiration.
- Gently resuspend the pellet in 5 mL supplemented Claycomb Medium, and add to the 10 mL of medium already in the T75 flask.
- Replace the medium with 15 mL of fresh supplemented Claycomb Medium 4 hours later (after cells have attached).
- Frozen vials should be thawed into a T25 flask.
Appearance–Clear orange-red solution
Endotoxin–Refer to Certificate of Analysis
Osmolality (as supplied)–Refer to Certificate of Analysis
pH (as supplied)–7.1 - 7.4
Sterility–No microbial growth detected