The overall goal of this procedure is to isolate Coob backer swarmer cells in order to establish a synchronized cell culture. This is accomplished by first growing a mixed culture of coob backer cells, and then briefly arresting growth by Resus suspending the cells in cold medium. The second step is to separate the swarmer cells through differential density centrifugation.
The next step is to resuspend the swarm cells in warm medium, which allows the cells to enter the cell cycle. Finally, the isolated swarm cells can be followed throughout the cell cycle by using biochemical assays or microscopy. The main advantage of this technique over other methods, such as the baby cell machine, is that cells can be isolated quickly and enlarge enough quantities for biochemical studies.
This method can help answer key questions in the cell biology field, such as what controls chromosome segregation and the asymmetric cell division cycle. To begin inoculate five milliliters of PYE medium with bacteria from a plate of colo backer CSIS strain, NA 1000. Grow the culture overnight at 28 degrees Celsius with shaking the following day, transfer 0.5 milliliters of the overnight culture to a flask containing 25 milliliters of M two G medium, and shake the flask at 28 degrees Celsius until the culture reaches an optical density between 0.5 and 0.6.
Next, dilute the culture into one liter of M two G medium to obtain the desired starting optical density and shake the flask at 28 degrees Celsius when the optical density of the culture reaches 0.5 pipette one microliter of the culture onto a slide, and then cover the liquid with a cover slip. Image the cells by phase microscopy to confirm the presence of rapidly swimming swarm cells. Transfer the culture to a sterile centrifuge tube and then spin the cells at 7, 000 times G for 15 minutes at four degrees Celsius.
Next, discard the loosely pelleted cells when pouring off the supernatant and add 180 milliliters of cold M two medium. Use a serological pipette to gently resuspend the cells. Then add 60 milliliters of cold colloidal silica solution to the cell suspension and mix well.
Pour the cell suspension into eight 30 milliliter tubes and centrifuge them at 6, 400 times G for 30 minutes. Set four degrees Celsius. Gently remove the tubes from the centrifuge and check the tube for two distinct bands.
The lower band contains the swarmer cells and pre divisional cells are in the top band. Next, carefully aspirate the top band and the liquid up to approximately one centimeter above the lower band. Use a paster pipette to remove the swarm band and transfer it to a clean tube.
Fill the tube with cold M two medium and then centrifuge the tube to wash away the colloidal silica. Next, carefully discard the supernatant and resuspend the cells in 20 milliliters of cold M two medium centrifuge. Again to pellet the cells, resuspend the pellets in 30 milliliters of cold M two medium, and then measure the optical density of the suspension.
Place one microliter of the cell suspension on a microscope slide and use phase imaging to check for swarm cells. Next, centrifuge the cells and resuspend them in M two G medium to an optical density between 0.3 and 0.4. Incubate the culture with shaking at 28 degrees Celsius for western plot or gene expression assays.
Remove one milliliter aliquots every 10 to 30 minutes for approximately 140 minutes until the optical density of the culture doubles. Use a tabletop centrifuge to spin down the cells for 30 seconds and then rapidly aspirate the medium flash. Freeze the cell pellets in liquid nitrogen and store them at negative 80 degrees Celsius for small scale synchrony inoculate five milliliters of M two G medium with NA 1000 cells and grow the cells overnight with shaking at 28 degrees Celsius the following day.
Dilute the culture in 15 milliliters of M two G medium and grow the cells until the optical density is between 0.5 and 0.6. Centrifuge the cells and then resuspend them in one milliliter of cold M two medium. Transfer the cells to a two milliliter micro centrifuge tube, pellet the cells by centrifugation and aspirate the sup natant.
Then place the tube on ice and resuspend the cell pellet in 900 microliters of cold M two medium. Next, add 900 microliters of cold PVP coated colloidal silica, and centrifuge the tube for 20 minutes. Carefully aspirate the top pre divisional cell band and then collect the bottom swarmer band into a new micro centrifuge tube.
Wash the swarmer cells two times in one milliliter of cold M two medium while centrifuging for three minutes between each wash right before the final spin. Place the cell suspension in a pre chilled one milliliter glass test tube and measure the optical density of the cells. Then transfer the cells to a micro fuge tube and centrifuge after centrifusion.
Reese, suspend the final cell pellet in Prewarm M two G medium at an optical density between 0.3 and 0.4 and shake the cells at 28 degrees Celsius. If microscopy is desired, place one microliter of the culture onto an M two G aeros pad at the appropriate time points. Differential density centrifugation of an asynchronous culture yields two bands of cells.
The bottom band contains swarmer cells, which have a higher density, and the top band contains stocked and pre divisional cells, which have a lower density. The optical density of the culture increases by approximately twofold during the course of the cell cycle. During this time, swarm cells differentiate into stocked cells, stocked cells then replicate and undergo asymmetric cell division.
With each division yielding a new swarmer cell and the parent stocked cell, a western blot for the cell cycle master regulator CTRA is a useful control to verify synchronization, CTRA blocks DNA replication in swarmer cells and is degraded upon DNA replication. CTRA is then synthesized later in the cell cycle preventing reinitiation of DNA replication and activating transcription of many developmental genes. This oscillating pattern of CTRA protein levels indicates a successful synchrony.
After watching this video, you should have a good understanding of how to synchronize callback or NA 1000 cells to investigate the bacterial cell cycle. This cell synchronization procedure can be done in one to two hours if performed correctly. While performing this procedure is important to remember to work quickly.
