The overall goal of this procedure is to induce hypoxia in cell culture. This is accomplished by first culturing cells in the presence of a chemical inducer of inducible factor one hypoxia by culturing the cells in a hypoxia incubator or chamber filled with a special gas mixture. Next hypoxia is detected by verifying the level of HIF one alpha expression in protein cell extracts, or by measuring its activity using a vector ENC coding for hi one alpha binding sequences.
Ultimately, results can be obtained that show an increase in HIF one alpha expression levels and activity through western blotting and luminescence detection. My name is Danny U.I'm a poster in the lab of Patrizia Inda. Today I will be demonstrating this procedure To induce hypoxia with cobalt chloride.
Begin by preparing a 25 millimolar stock solution of cobalt chloride in sterile double distilled water. Add the solution to cells and culture at a final concentration of 100.Micromolar. Incubate the cells for 24 hours at 37 degrees Celsius and 5%carbon dioxide to induce hypoxia.
Using a modular incubator chamber, prepare at least two identical cell cultures. However, a triplicate of each condition is recommended. Open the modular incubator chamber by first opening the two white plastic clamps located on the tubes attached to the chamber, and remove the lid and trays.
Place the cell culture in the hypoxic chamber with a Petri dish containing sterile water in the chamber to provide adequate humidification of the cultures. Place the second cell culture in Normoxia as a control to induce hypoxia. Attach the tubing to a hypoxia tank containing a 1%oxygen gas mixture.
If a flow meter is connected to your tank, the chamber will be directly connected to it. We use a flow meter incorporated in our regulator to remove all oxygen present in the chamber and in the medium. Flush the chamber by opening the gas tank at a flow rate of 20 liters per minute for four to seven minutes.
Then quickly turn off the gas flow and completely close the chamber. By closing both white clamps. Return the chamber to a conventional incubator for the desired period of time.
If using large cultures, allow the medium and the cultures to DGAs for one to two hours, and then repeat the flush to evaluate hypoxia. Using HIF one alpha detection by western blot, reopen the chamber as demonstrated earlier and immediately place the cultures on ice. Lice the hypoxia treated and non-treated cells with 5%SDS solution.
Then transfer the lysates to tubes and sonicate to extract the proteins, spin down the cell debris and collect the sate. Next, measure the protein concentration using A BCA kit. Then prepare a gel sample by adding loading buffer and heating at 95 degrees Celsius for five minutes.
Separate the proteins on an 8%SDS poly acrylamide gel and transfer them to a nitrocellulose membrane. Block the membrane in PBS containing 5%non-fat dry milk and 0.1%tween 20 at room temperature for one hour or at four degrees Celsius overnight on a shaker. Incubate the blot overnight with an anti HIF one alpha primary monoclonal antibody in the same buffer at four degrees Celsius.
The next day. Wash the blot three times for five minutes each in PBS containing 0.1%tween 20 at room temperature, then incubate with an HRP secondary antibody in PBS plus 0.1%tween 20 for 45 minutes. Wash three times for five minutes each in PBS containing 0.1%tween 20 at room temperature.
Then incubate the membrane in chemiluminescent detection solutions and expose it to x-ray film to measure luciferase activity. Remove the growth medium from the cultured cells. Rinse the cells in PBS.
Then add 400 microliters of one x lysis reagent into the culture dish. Scrape the attached cells and transfer them to a micro tube. Pellet the debris by brief centrifugation.
Mix 20 microliters of cell lysate supernatant with a hundred microliters of luciferase assay reagent, and measure the luminescence using a luminometer. K 5 62 cells were cultured in normoxia or hypoxia for 48 hours and analyzed by western blot using an antibody specific for H one alpha. An antibody specific for beta actin was used for the loading control.
The results show an increase in HI F1 alpha in hypoxic cells. HRE Luciferase modified 2 93 cells were cultured in hypoxia or normoxia for 48 hours, and then laced to detect a luciferase signal using a luciferase assay kit and luminometer here. An increase in HIF one alpha activity was also detected in hypoxic cells.
The results are expressed as relative luminescence. Units Don't forget that working with precise gas can be extremely hazardous and precautions such as securing the oxygen tank to the wall or bench properly opening and closing the oxygen tank, as well as controlling the pressure should always be taken while performing this procedure.
