Lab Protocols

Procedure: Utilize the following mix components for all experiments. This protocol has been optimized for transfection of neonatal rat cardiac myocytes. For those experiments where more transfection mix is needed, simply use a multiple of the reagents described below: For cells on 24 well plates, combine equal amounts of the plasmid in question and normalization signal with L7RH-beta-Gal plasmid. Add 40 μl per well immediately after plating (20 μl each of the luciferase plasmid with 20 μl of the beta gal mix). All mixing (except that which requires vortexing) should be done in the sterile cell culture hood. Total volume 400 μl 600 μl 800 μl Sterile 2.5 M CaCl 2 25 μl 37.5 μl 50 μl Plasmid 10 μg 15 μg 20 μl Sterile H 2 0 Total of 200 μl when added to above. Bring to total of 300 μl with sterile H 2 0 Bring total to 400 μl with H 2 0  Combine the above three reagents in a hood using a sterile 1.5 ml Eppendorf tube. Mix gently. Do not vortex. Sterile 2X HBS (hepes buffered sa...

Procedure Day 0 cell culture: Grow 3T3-L1 cells in high glucose DMEM + 10% Calf Serum + P/S/F in 6-well plates. After cell have reached complete confluent, replace growth media with stimulation media containing: High glucose DMEM: 10% FCS, P/S/F 1 µl / ml of Insulin (10 mg/ml, 1000x in HEPES, final 10 µg/ml) 1 µl / ml IBMX (3-isobutyl-1-methylxanthine, 500 mM stock, 1000x), final 0.5 mM 0.1 µl / ml Dexamethasone (10000X in ethanol, final 1 µM). Day 3 Replace with fresh media containing Insulin only, no IBMX, no Dexamethasone.  Change the media on day 6.  Cells will be differentiated on day 7-9. Day 6 Cells will be cultured in serum-free DMEM for 2 hr ± treatments Incubate cells  in 1 ml of KRH with 100 nM insulin for 20 min ± treatments. Add 1 µCi of 2-deoxy-D-[2,6-3H]-glucose (50 µl in volume) to each well for 10 min. Wash cells with KRH buffer 3X. Lyse cells in 1 ml of RIPA buffer.  Count 300 µl. KRH buffer: 25 mM HEPES-NaOH [pH 7.4] 1...

Procedure describing a suggested protocol for sarcomeric actinin, ANP, ER and ERb fluorescent staining of cardiomyocytes View a pdf version of this page   Anti-sarcomeric – α actinin and ANP Staining Fix cells as usual in 3.7% paraformaldehyde in PBS. Rinse cells twice with PBS after fixation. Permeabilize and block in 2% FBS / 2% BSA in PBS with 0.1% NP40 for 45 min. Treat cells with antibodies for α-actinin eg.  alpha sarcomeric actin antibody [alpha Sr-1] (ab28052)  and ANP diluted in the blocking buffer as above for 1 hour. Wash x3 in PBS for 5 min each. Add secondary ab in the same blocking buffer (for green ab: CY2 or fluoroscein labeled abs, use 1/200 dilution of secondary ab and for CY3 or Rhodamine, use 1/1000 dilution for 45 min (though these will require some optimization) Wash in PBS x3 Add DAPI solution to coverslips for 15 min. Wash x2 in PBS. Add mounting solution and coverslip. Image cells within 2 days. The α-actinin stain is stable for quite so...

Apoptosis or programmed cell death is defined as " a mechanism of cellular suicide which occurs after sufficient cellular damage ". Apoptosis is first characterized by a change in the refractive index of the cell followed by cytoplasmic shrinkage and nuclear condensation. The cell membrane begins to show blebs and eventually these blebs separate from the dying cell and form "apoptotic bodies". Apoptotic cells also cease to maintain phospholipid asymmetry in the cell membrane, and phosphotidylserine appears on the outer leaflet. The mitochondrial outer membrane also undergoes changes that include loss of its electrochemical gradient, and substances like cytochrome c leak into the cytoplasm. Finally, adjacent cells or macrophages phagocytose apoptotic bodies and the dying cell. The apoptotic cell does not provoke an inflammatory response, and only individual cells are affectedby apoptosis in vivo . The mechanisms of apoptosis Apoptosis can be induced in response to ...

Procedure: Pellet cells Lyse cells in 0.5 ml detergent buffer: 10 mm Tris (pH 7.4), 5 mm EDTA, 0.2% Triton  Vortex  Incubate on ice for 30 min. Centrifuge at 27000 g for 30 minutes  Divide supernatants into 2-250 μl aliquots  Add 50 ul ice cold 5 M NaCl to each followed by vortexing  Precipitate DNA: Add 0.6 ml 100% EtOH and 150 μl 3 M Na acetate, pH 5.2 place at –80oC freezer for 1 hr. Centrifuge 20,000 g for 20 min and pool DNA extracts by re-dissolving by adding 400 μl total) of extraction buffer (10 mM Tris and 5 mm EDTA).  Add 2 μl (10 mg/ml) DNase free RNase. Incubate for 5 hr at 37 o C.  Add 25 μl Proteinase K at 20 mg/ml and 40 μl of buffer (100 mm Tris, pH 8.0, 100 mM EDTA, 250 mM NaCl. Incubate overnight at 65 o C.  Extract DNA with phenol, choloform, isoamyl alcohol and precipitate with EtOH. From Kotamraju et al JBC, 2000 DNA Fragmentation: A distinctive feature of apoptosis at the biochemical level is DNA fragmentation. T...

Introduction Caspases are essential in cells for apoptosis, one of the main types of programmed cell death in development and most other stages of adult life, and have been termed "executioner" proteins for their roles in the cell. Caspases were first implicated in apoptosis when CED-3, a protein required for programmed cell death in Caenorhabditis elegans, was found to have close homology with the mammalian interleukin-1 -converting enzyme (ICE or caspase 1) and that over-expression of ICE induced apoptosis. Failure of apoptosis is one of the main causes of tumour development and autoimmune diseases. This coupled with the unwanted apoptosis that occurs with ischaemia or Alzheimer's disease, has raised interest in caspases as potential therapeutic targets. Caspases are enzymes known as proteases, which play essential roles in apoptosis and inflammation. As proteases, they are enzymes that cleave other proteins. They are called cysteine proteases, because they use a cyste...

Apoptosis may be induced in experimental systems through a variety of methods. In general, they can be divided into 2 categories: a) biological induction; and b) chemical induction. A) Biological induction of apoptosis Activation of either Fas or TNF-receptors by the respective ligands or by cross-linking with agonist antibody induces apoptosis of Fas- or TNF receptor-bearing cells. Below is general protocol used to induce apoptosis using anti-Fas receptor (CD95) mAb in Jurkat Cells. 1. Grow Jurkat cells in RPMI-1640 medium containing 10% fetal bovine serum in a humidified, 5% CO 2 incubator at 37°C. 2. Suspend the cells in fresh medium at a concentration of 1 × 105 cells/ml. After two to three days of incubation in a 37°C, 5% CO 2 incubator, harvest the cells by centrifugation at 300–350 × g for 5 mins. 3. Resuspend cells in fresh medium to 5 × 105 cells/ml and add CD95 mAb to a final concentration of 0.05 – 0.1 μg/ml. Incubate for 3–6 hours in a 37°C incubator. As a negativ...

The appearance of phosphatidylserine (PS) residues (normally hidden within the plasma membrane) on the surface of the cell is an early parameter of apoptosis, which can also be used to detect and measure apoptosis. During apoptosis, PS is translocated from the cytoplasmic face of the plasma membrane to the cell surface. Annexin V has a strong, Ca2+-dependent affinity for PS and therefore can be used as a probe for detecting apoptosis. Example protocol -  Protocol for Annexin V-FITC Apoptosis Detection Kit (ab14085) : A) Incubation of cells with Annexin V-FITC Induce apoptosis by desired method. Collect 1-5 x 10 5 cells by centrifugation. Resuspend cells in 500 μl of 1X Binding Buffer. Add 5 μl of Annexin V-FITC and 5 μl of propidium iodide (PI, optional.) Incubate at room temperature for 5 min in the dark. Proceed to B or C below depending on method of analysis. B) Quantification by flow cytometry Analyze Annexin V-FITC binding by flow cytometry (Ex = 488 nm; Em = 530 nm) ...