Analysis of lipid metabolism kinetics by pulse-chase in living cells (Thiele lab)

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'''Analysis of lipid metabolism kinetics&nbsp; by pulse-chase in living cells (Thiele lab)'''<br><br>Rationale: Using this protocol, the dynamics of cellular lipid metabolism can be analyzed. For labeling the metabolic pool, the protocol uses a radioactive fatty acid, which allows parallel study of turnover of both glycerophospholipids and triacylglycerol. Radioactive metabolites are separated by TLC, followed by detection using fluorography. By variation of the labeling compound, for example by use of radioactive sphingosine, choline, serine or sugars, other lipids or specific subgroups can be followed. Instead of radioactive compounds, also fluorescent analogues or stable-isotope labeled compounds can be used. In this case, subsequent analysis is performed by mass spectrometry.<br><br>'''Step 1''':<br>Grow cell lines of interest in the appropriate culture medium until confluency in 6 cm or 10 cm dishes.<br><br>'''Step 2:'''<br>Prepare the following solutions:<br><br>'''Pulse medium''': Culture medium + 50 µCi/ml of 3H oleic acid (spec. act. 30-50 Ci/mmol). For 10 cm dishes, you will need 5 ml per dish per time-point of the analysis. Warm to 37 degree Celsius.<br><br>'''Chase medium''': Culture medium of your cell line. Warm to 37 degree Celsius.<br><br>'''Wash solution''': PBS + 10 mg/ml fatty acid free BSA. Warm to 37 degree Celsius.<br><br>'''Stop solution:''' PBS + 10 mg/ml fatty acid free BSA. Cool on ice.<br><br>'''PBS'''. Cool on ice<br><br>'''Methanol/Chloroform 2/1'''<br>'''<br>Step 3''':<br>Remove the culture medium from a dish and add 5 ml '''Pulse medium'''. Incubate in a waterbath at 37 degrees Celsius. Pulse time counts from the addition of Pulse medium until the addition of wash solution in Step 4. For labeling with a radioactive fatty acid, a pulse time of 1 min is recommended.<br><br>'''Step 4''': Remove the Pulse medium by aspiration and add 8 ml '''Wash solution'''. Gently move the dish to distribute the solution. Remove the Wash solution. Work quick, but take care to thoroughly and completely exchange the media. <br><br>'''Step 5''':&nbsp; <br>Add the '''chase medium''' (8 ml/10 cm dish), put a lid onto the dish and incubate at 37 degrees Celsius. Reasonable chase times for fatty acid labeling should cover 1-60 min.<br><br>'''Step 6''': <br>Remove the Chase medium and add 10 ml of ice-cold '''Stop solution'''. From here on, all steps are performed on ice and with ice-cold solutions. Gently move the plate to distribute the solution and remove it. Repeat once with Stop solution.<br><br>'''Step 7:''' <br>Perform a final wash with 8 ml ice-cold '''PBS'''. Completely remove the PBS, add 1 ml ice cold PBS and scrape the cells using a soft cell scraper (see below).<br><br>'''Step 8:'''<br>Transfer the cell suspension into a tube containing 4 ml '''chloroform/methanol 1/2'''. Now the metabolism is stopped. For subsequent lipid extraction and analysis by TLC, see the protocols “Lipid extraction for TLC (Chloroform-Methanol, Thiele lab)” and “Thin layer chromatography (Thiele Lab)”.<br>Spray the developed TLC plate with a scintillant (we use Lumasave from Lumac) and expose to X-ray films (we use Hyperfilm MP from Amersham. Other high sensitivity films for autoradiography such as Kodak X-OMAT AR can be used. Do not use a film designed for ECL detection) at -80 degrees Celsius until the signals are sufficient but not over-saturated. This takes usually 1-5 days. Scan the films with a good scanner and quantify the spots. Take care to remove any non-linear setting and any threshold setting in the scanning and analysis software.<br><br><br>'''Comments:''' The success of a pulse-chase experiment depends on (i) controlling temperature during pulse and chase and (ii) on speed and precision in handling. You need a cell culture incubator, one large waterbath for cell culture dishes and one waterbath for media bottles on your bench or directly next to it. Prewarmed solutions (Pulse medium, chase medium, and the wash medium between pulse and chase) should be pre-portioned into 15 ml tubes and poured onto the plates, since pipetting takes too long and cools the solutions. Before doing a radioactive experiment on precious cells, optimize the handling with non-radioactive solutions and empty dishes.<br>'''<br>Time table:''' You can process plates one by one, in which case the experiment takes very long because of long chase times. It is advisable to start with the pulses of the long chases and process plates for shorter chase times during the long chases. For that you need to prepare an exact time-table (example see below). Make sure that you can work without any interruption.<br><br>Plate number&nbsp;&nbsp;&nbsp; Pulse – Chase duration&nbsp;&nbsp;&nbsp; Pulse starts at minute&nbsp;&nbsp;&nbsp; Chase starts at minute&nbsp;&nbsp;&nbsp; Finish at minute<br>1&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 1 + 0&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 45&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 46&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 46<br>2&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 1 + 1&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 40&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 41&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 42<br>3&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 1 + 2&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 34&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 35&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 37<br>4&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 1 + 3&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 28&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 29&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 32<br>5&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 1 + 5&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 16&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 17&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 22<br>6&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 1 + 10&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp; 13&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 14&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 24<br>7&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 1 + 15&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 10&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 11&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 26<br>8&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 1 + 30&nbsp;&nbsp; &nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp; 7&nbsp;&nbsp; &nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp; 8&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp; 38<br>9&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 1 + 60&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 4&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp; 5&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp; 65<br>10&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp; 1 + 90&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 1&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp; 2&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 92<br><br>'''Anticipated result:''' Using a radioactive fatty acid, you should see disappearance of the free fatty acid during the 1 min chase. PA will disappear with a half life of 2 min. DAG will be formed and peak at about 3 min, than decay with 5 min half life. PC, other phospholipids and TG steadily increase and reach plateaus after about 30 min. <br><br><br>'''Soft cell scraper:''' Most commercial single-use cell scrapers are made from a relatively hard plastic, which works very poorly. To get a good, efficient scraper, take a stopper made from white silicon rubber (diameter 1-4 cm, according to the desired size of the scraper), and cut with a sharp knife a 2-3 mm thick round slice. This slice you cut into two half discs. Take care to get straight edges. This is a great cell scraper, which you can hold either with a paper clip or connect to a plastic pipette (by a hole that you punch at the round side)&nbsp; <br><br>  
'''Analysis of lipid metabolism kinetics&nbsp; by pulse-chase in living cells (Thiele lab)'''<br><br>Rationale: Using this protocol, the dynamics of cellular lipid metabolism can be analyzed. For labeling the metabolic pool, the protocol uses a radioactive fatty acid, which allows parallel study of turnover of both glycerophospholipids and triacylglycerol. Radioactive metabolites are separated by TLC, followed by detection using fluorography. By variation of the labeling compound, for example by use of radioactive sphingosine, choline, serine or sugars, other lipids or specific subgroups can be followed. Instead of radioactive compounds, also fluorescent analogues or stable-isotope labeled compounds can be used. In this case, subsequent analysis is performed by mass spectrometry.<br><br>'''Step 1''':<br>Grow cell lines of interest in the appropriate culture medium until confluency in 6 cm or 10 cm dishes.<br><br>'''Step 2:'''<br>Prepare the following solutions:<br><br>'''Pulse medium''': Culture medium + 50 µCi/ml of 3H oleic acid (spec. act. 30-50 Ci/mmol). For 10 cm dishes, you will need 5 ml per dish per time-point of the analysis. Warm to 37 degree Celsius.<br><br>'''Chase medium''': Culture medium of your cell line. Warm to 37 degree Celsius.<br><br>'''Wash solution''': PBS + 10 mg/ml fatty acid free BSA. Warm to 37 degree Celsius.<br><br>'''Stop solution:''' PBS + 10 mg/ml fatty acid free BSA. Cool on ice.<br><br>'''PBS'''. Cool on ice<br><br>'''Methanol/Chloroform 2/1'''<br>'''<br>Step 3''':<br>Remove the culture medium from a dish and add 5 ml '''Pulse medium'''. Incubate in a waterbath at 37 degrees Celsius. Pulse time counts from the addition of Pulse medium until the addition of wash solution in Step 4. For labeling with a radioactive fatty acid, a pulse time of 1 min is recommended.<br><br>'''Step 4''': Remove the Pulse medium by aspiration and add 8 ml '''Wash solution'''. Gently move the dish to distribute the solution. Remove the Wash solution. Work quick, but take care to thoroughly and completely exchange the media. <br><br>'''Step 5''':&nbsp; <br>Add the '''chase medium''' (8 ml/10 cm dish), put a lid onto the dish and incubate at 37 degrees Celsius. Reasonable chase times for fatty acid labeling should cover 1-60 min.<br><br>'''Step 6''': <br>Remove the Chase medium and add 10 ml of ice-cold '''Stop solution'''. From here on, all steps are performed on ice and with ice-cold solutions. Gently move the plate to distribute the solution and remove it. Repeat once with Stop solution.<br><br>'''Step 7:''' <br>Perform a final wash with 8 ml ice-cold '''PBS'''. Completely remove the PBS, add 1 ml ice cold PBS and scrape the cells using a soft cell scraper (see below).<br><br>'''Step 8:'''<br>Transfer the cell suspension into a tube containing 4 ml '''chloroform/methanol 1/2'''. Now the metabolism is stopped. For subsequent lipid extraction and analysis by TLC, see the protocols “Lipid extraction for TLC (Chloroform-Methanol, Thiele lab)” and “Thin layer chromatography (Thiele Lab)”.<br>Spray the developed TLC plate with a scintillant (we use Lumasave from Lumac) and expose to X-ray films (we use Hyperfilm MP from Amersham. Other high sensitivity films for autoradiography such as Kodak X-OMAT AR can be used. Do not use a film designed for ECL detection) at -80 degrees Celsius until the signals are sufficient but not over-saturated. This takes usually 1-5 days. Scan the films with a good scanner and quantify the spots. Take care to remove any non-linear setting and any threshold setting in the scanning and analysis software.<br><br><br>'''Comments:''' The success of a pulse-chase experiment depends on (i) controlling temperature during pulse and chase and (ii) on speed and precision in handling. You need a cell culture incubator, one large waterbath for cell culture dishes and one waterbath for media bottles on your bench or directly next to it. Prewarmed solutions (Pulse medium, chase medium, and the wash medium between pulse and chase) should be pre-portioned into 15 ml tubes and poured onto the plates, since pipetting takes too long and cools the solutions. Before doing a radioactive experiment on precious cells, optimize the handling with non-radioactive solutions and empty dishes.<br>'''<br>Time table:''' You can process plates one by one, in which case the experiment takes very long because of long chase times. It is advisable to start with the pulses of the long chases and process plates for shorter chase times during the long chases. For that you need to prepare an exact time-table (example see below). Make sure that you can work without any interruption.<br><br>Plate number&nbsp;&nbsp;&nbsp; Pulse – Chase duration&nbsp;&nbsp;&nbsp; Pulse starts at minute&nbsp;&nbsp;&nbsp; Chase starts at minute&nbsp;&nbsp;&nbsp; Finish at minute<br>1&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 1 + 0&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 45&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 46&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 46<br>2&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 1 + 1&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 40&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 41&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 42<br>3&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 1 + 2&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 34&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 35&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 37<br>4&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 1 + 3&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 28&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 29&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 32<br>5&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 1 + 5&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 16&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 17&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 22<br>6&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 1 + 10&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp; 13&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 14&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 24<br>7&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 1 + 15&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 10&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 11&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 26<br>8&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 1 + 30&nbsp;&nbsp; &nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp; 7&nbsp;&nbsp; &nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp; 8&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp; 38<br>9&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 1 + 60&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 4&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp; 5&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp; 65<br>10&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp; 1 + 90&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; 1&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp; 2&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 92<br><br>'''Anticipated result:''' Using a radioactive fatty acid, you should see disappearance of the free fatty acid during the 1 min chase. PA will disappear with a half life of 2 min. DAG will be formed and peak at about 3 min, than decay with 5 min half life. PC, other phospholipids and TG steadily increase and reach plateaus after about 30 min. <br><br><br>'''Soft cell scraper:''' Most commercial single-use cell scrapers are made from a relatively hard plastic, which works very poorly. To get a good, efficient scraper, take a stopper made from white silicon rubber (diameter 1-4 cm, according to the desired size of the scraper), and cut with a sharp knife a 2-3 mm thick round slice. This slice you cut into two half discs. Take care to get straight edges. This is a great cell scraper, which you can hold either with a paper clip or connect to a plastic pipette (by a hole that you punch at the round side)&nbsp; <br><br>  
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[[Category:Lipid_metabolism_analysis]]
+
[[Category:Lipid_metabolism_analysis]] [[Category:Radioactive_labeling]]

Current revision

Analysis of lipid metabolism kinetics  by pulse-chase in living cells (Thiele lab)

Rationale: Using this protocol, the dynamics of cellular lipid metabolism can be analyzed. For labeling the metabolic pool, the protocol uses a radioactive fatty acid, which allows parallel study of turnover of both glycerophospholipids and triacylglycerol. Radioactive metabolites are separated by TLC, followed by detection using fluorography. By variation of the labeling compound, for example by use of radioactive sphingosine, choline, serine or sugars, other lipids or specific subgroups can be followed. Instead of radioactive compounds, also fluorescent analogues or stable-isotope labeled compounds can be used. In this case, subsequent analysis is performed by mass spectrometry.

Step 1:
Grow cell lines of interest in the appropriate culture medium until confluency in 6 cm or 10 cm dishes.

Step 2:
Prepare the following solutions:

Pulse medium: Culture medium + 50 µCi/ml of 3H oleic acid (spec. act. 30-50 Ci/mmol). For 10 cm dishes, you will need 5 ml per dish per time-point of the analysis. Warm to 37 degree Celsius.

Chase medium: Culture medium of your cell line. Warm to 37 degree Celsius.

Wash solution: PBS + 10 mg/ml fatty acid free BSA. Warm to 37 degree Celsius.

Stop solution: PBS + 10 mg/ml fatty acid free BSA. Cool on ice.

PBS. Cool on ice

Methanol/Chloroform 2/1

Step 3
:
Remove the culture medium from a dish and add 5 ml Pulse medium. Incubate in a waterbath at 37 degrees Celsius. Pulse time counts from the addition of Pulse medium until the addition of wash solution in Step 4. For labeling with a radioactive fatty acid, a pulse time of 1 min is recommended.

Step 4: Remove the Pulse medium by aspiration and add 8 ml Wash solution. Gently move the dish to distribute the solution. Remove the Wash solution. Work quick, but take care to thoroughly and completely exchange the media.

Step 5
Add the chase medium (8 ml/10 cm dish), put a lid onto the dish and incubate at 37 degrees Celsius. Reasonable chase times for fatty acid labeling should cover 1-60 min.

Step 6:
Remove the Chase medium and add 10 ml of ice-cold Stop solution. From here on, all steps are performed on ice and with ice-cold solutions. Gently move the plate to distribute the solution and remove it. Repeat once with Stop solution.

Step 7:
Perform a final wash with 8 ml ice-cold PBS. Completely remove the PBS, add 1 ml ice cold PBS and scrape the cells using a soft cell scraper (see below).

Step 8:
Transfer the cell suspension into a tube containing 4 ml chloroform/methanol 1/2. Now the metabolism is stopped. For subsequent lipid extraction and analysis by TLC, see the protocols “Lipid extraction for TLC (Chloroform-Methanol, Thiele lab)” and “Thin layer chromatography (Thiele Lab)”.
Spray the developed TLC plate with a scintillant (we use Lumasave from Lumac) and expose to X-ray films (we use Hyperfilm MP from Amersham. Other high sensitivity films for autoradiography such as Kodak X-OMAT AR can be used. Do not use a film designed for ECL detection) at -80 degrees Celsius until the signals are sufficient but not over-saturated. This takes usually 1-5 days. Scan the films with a good scanner and quantify the spots. Take care to remove any non-linear setting and any threshold setting in the scanning and analysis software.


Comments: The success of a pulse-chase experiment depends on (i) controlling temperature during pulse and chase and (ii) on speed and precision in handling. You need a cell culture incubator, one large waterbath for cell culture dishes and one waterbath for media bottles on your bench or directly next to it. Prewarmed solutions (Pulse medium, chase medium, and the wash medium between pulse and chase) should be pre-portioned into 15 ml tubes and poured onto the plates, since pipetting takes too long and cools the solutions. Before doing a radioactive experiment on precious cells, optimize the handling with non-radioactive solutions and empty dishes.

Time table:
You can process plates one by one, in which case the experiment takes very long because of long chase times. It is advisable to start with the pulses of the long chases and process plates for shorter chase times during the long chases. For that you need to prepare an exact time-table (example see below). Make sure that you can work without any interruption.

Plate number    Pulse – Chase duration    Pulse starts at minute    Chase starts at minute    Finish at minute
1                                1 + 0                                    45                                46                            46
2                                1 + 1                                    40                                41                            42
3                                1 + 2                                    34                                35                            37
4                                1 + 3                                    28                                29                            32
5                                1 + 5                                    16                                17                            22
6                                1 + 10                                  13                                14                            24
7                                1 + 15                                  10                                11                            26
8                                1 + 30                                    7                                 8                             38
9                                1 + 60                                    4                                 5                             65
10                              1 + 90                                    1                                 2                              92

Anticipated result: Using a radioactive fatty acid, you should see disappearance of the free fatty acid during the 1 min chase. PA will disappear with a half life of 2 min. DAG will be formed and peak at about 3 min, than decay with 5 min half life. PC, other phospholipids and TG steadily increase and reach plateaus after about 30 min.


Soft cell scraper: Most commercial single-use cell scrapers are made from a relatively hard plastic, which works very poorly. To get a good, efficient scraper, take a stopper made from white silicon rubber (diameter 1-4 cm, according to the desired size of the scraper), and cut with a sharp knife a 2-3 mm thick round slice. This slice you cut into two half discs. Take care to get straight edges. This is a great cell scraper, which you can hold either with a paper clip or connect to a plastic pipette (by a hole that you punch at the round side) 

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