The overall objective of this study is to obtain a Time-Lapse 2D Imaging of Phagocytic activity in M1 Macrophages 4T1 Mouse Mammary Carcinomas Cells in Co-cultures. The life cells co-cultures model was established and observed using a combination of fluorescent and differential interference contrast microscopy. The assessment of phagocytosis activity by macrophages were image;using imaging software to double up multi point time lapse video, prior to atesting our incubation.
The matter that we conducted about to fulfill the needs and benefits of the honing in the behavior of macrophages and its phagocytic activity with cancer cells. Specifically in the study is mouse mammary carcinoma. The first part of this study begins with live cells co-culture model of 4T1 mouse mammary carcinomas cells and RAW 2647 mouse macrophages.
Begin the experiment by culturing 41 mouse mammary carcinomas cells and RAW 2647 mouse macrophages. First, thaw a vial of 4T1 and RAW 2647 cells. Dilute the thawed cells in 10 millimeter in a 15 mill conical tube.
Next, centrifuge the cells at 660 g force for 5 minutes to obtain cell pellet. Carefully aspirate the media, re-suspend the cells in 8 mill of complete media and transfer the cells into tissue culture flask. Incubate the cells at 37 degrees Celsius with 5 percent carbon dioxide.
After one week of culturing under adherent conditions, monitor the flask daily and supplement with atmil complete media as needed. The next step continue with the immunostaining of M1 polarized RAW 2647 macrophages. This step is to ensure a complete polarization of M1 macrophages.
As the confluency of RAW 2647 has reached 70 to 80 percent discard the culture media and gently rinse twice with PBS. Prepare the macrophages for collectioned by gently dislodge the adherent cells using a cell scrapper and transfer into a 50 mill conical tube. Count cells by using hemocytometer and prepare a cell suspensioned at a density of 100, 000 cells per dish by adding the remaining solution to the sitting density.
Sit 2 mill of macrophages suspension into two imaging dishes. Incubate the cells 2 to 3 hours at 37 degrees celsius with 5 percent carbon dioxide to allow cell adherence. Prepare M1 polarization media by adding 100 nilligram per mill LPS and 20 nilligram per mill interferon-gamma into a complete medium supplemented with 10 percent FBS.
Discard the cultures supernatant from macrophages incubated before and carefully wash the monolayers in the dish twice using PBS. Add 2 mill of M1 polarization media into one of the matching dishes and allow the dura cells to infuse into M1 macrophages finnatype. Before immunostaining, fix RAW and M1 macrophages cells using 2 mill of 4 percent paraformaldehyde and incubate for 50 minute in room temperature.
Remove the supernatant and wash the cells monolayer using PBS, at least three times. Quench with 2 mill of 50 millimeter ammonium chloride in PBS and incubate for 50 minute in room temperature. Permeabilize using 0.3 percent Triton X-100 in PBS for 50 minute in room temperature.
Blocking step using 10 percent FBS for 30 minutes in PBS room temperature. Incubate with anti-iNOS antibody conjugated FITC in PBS and one percent FBS overnight at 4 degrees Celsius. Incubate 1 mill of DAPI into imaging dishes for 10 minute at 37 degrees Celsius in dark condition by covering with aluminum foil.
Wash cells using PBS at least 3 times and add 1 mill of DMEM into matching dishes and ready for fluorescents imaging. The next step proceed with seeding of 4T1 mouse mammary carcinomas cells. As the confluency of 4T1 cells reach 70 to 80 percent discard the culture media and gentley rinse twice with PBS.
Add 1 mill of pre-warmed trypsin to de-associate the cells and incubating for up to 20 minutes at 37 degrees Celsius. Once the cells appear de-attached, transfer the de-attached cells to a 15 mill conical tube. And add 2 mill of pre-warmed complete medium to inactivate the tube seen.
Centrifuge at 600 g force for 5 minute to obtain a cell pellet. Remove the supernatant and re-suspend pellet in 10 mill prerun sampling DMEM medium. Count the cells by using hemocytometer and seed 100, 000 cells into mill DMEM.
Into imaging these with TC treated. The next step is labeling living 4T1 mouse mammary carcinomas cells using CFSE staining. First, remove the existing media from the imaging dish that previously seeded with 4T1 cells.
Prepare CFSE staining solution by diluting CFSE staining in 1 mill PBS to make 5 macrmal out working consentrationed. Add 1 mill of CFSE staining solution into imaging dish to incubate cells for 20 minute at room temperature or 37 degrees Celsius in the dark. Add an equal of DMEM to the cells plus staining solution and allow to seed for 5 minute.
The next step is to seed RAW2647 mouse macrophages and co-culture with 4T1. After CFSE leveling of 4T1 cells, wash the imaging dish twice, using PBS. Seed 2 mill of macrophages suspension into 4T1 imaging dish.
The next step is to polarize RAW 4627 macrophages into M1 finnatype. Add 2 mill of M1 polarization media into the imagine dish and allow the RAW cells to infuse into M1 macrophage finnatype. The second part of the study is time-lapse assessment of M1 macrophages phagocyted 4T1 cells.
The next step includes live-cell video microscopy phagocytosis assessment. First, switch on the microscope. Set up temperature at 37 degrees Celsius.
And 5 percent carbon dioxide gas. Position the co-culture cells in the center of the stage and gently screw on the top chamber. Use the joystick to motorize the stage by selecting different position to be captured.
There are many factors need to be considered while performing live-cell imaging. To obtain a pretty small video. It requires optimization of imaging exposure, time measurement and also automating focus correction.
In case the video is over exposed, incorrect time interval, and also out of focus, will cost the video to be unusable. Repetition of experiment need to be done if one of these factors been detected. Figure one shows immunofluorescences turning with anti-iNOS in green.
Nuclear marker DAPI in blue. And enhanced versions of most images. Movie 1 is a representative live-cell movie of understanding M1 macrophages and 4T1.
CFSE stained most mammary carcinomas cells co-cultures captured using multi-channel acquisition of florescent and the IC microscopy. Movie 2 live-cell video microscopy movies from multi-point in a single conjugal dish. Showing phagocytosis of 4T1 by induced M1 macrophages.
Images were captured at 50 minute intervals for 13 hour. Movie 3 live-cell video microscopy movie, of a single coordinate point chosen to show an in depth visualization of the phagocytosis of 4T1 by M1 Macrophages. Red arrows shows to M1 macrophages, yellow circle shows that a number of 4T1 cells quench fluorene and goffman of 4T1 cells.
M1 macrophages move-ology describe as porocytoplasm were as 4T1 mouse memory carcinomas have an epithelium morphology. Macrophages can be seen moving to add 4T1 cells to establish cell-to-cells contact. Which is then followed by the uptake of 4T1 cells per M1 macrophages.
After watching this video you should have a good understanding on how to perform live-cell video microscopy experiment;with 4T1 mouse mammary carcinoma and RAW 2647 merad macrophages. You should also able to set up over night co-cultures of both cell types to perform phagocytosis essay. You should also understand on how the co-cultuers of 4T1 with LPS and interferon-gamma macrophages.
