I am performing Annexin V FITC assay for adherent cells. According to the protocol described, the treated cells were trypsinised and collected, followed by washing them twice with cold PBS. The cell pellet was suspended in 1x binding buffer at a concentration of 1 x 10^6 cells/ml. 100ul of the solution was transferred to a 5ml culture tube and stained with the dyes, followed by 15mins incubation in the dark. 400ul of 1x binding buffer was topped up before reading.
I would like to know that when we are suppose to count the cells before suspending them in 1x binding buffer? Is it after washing twice with PBS or after the first time of washing?
Next, in order to count the cells, some solution (PBS or binding buffer for example) will need to be added to the cell pellet in order to aliquot for counting under a hemocytometer. After determination of the volume with the desired cell number ( 1 x 10^6 cells), the cells were centrifuged again to get the cell pellet, and the cell pellet was suspended in 1x binding buffer before transferring 100ul out to a new tube. Is this the correct way to get 1 x 10^6 cells?
How do the cell number normally be determined in Annexin V assay? Will the cell number affect the results? Are there any better way to do the counting of cells? I need some advise from all of you. Very much appreciation. :)
Dear Han,
I would suggest and second Harald Kühnel
method. After trypsinization, wash your cells once with PBS and then re-suspend in the 1mL PBS. Now take 10 uL of this cell suspension, mix it in 90 uL of PBS and count your cells. calculate per mL cells according to the formula.Then proceed for the second wash and discard supernatent, followed by the Annexin/PI staining protocol.
Hope this is helpful.
Hello, what I can suggest is not doing counting since you can know the number of your cells through the FACS lecture because I think that a counting after trypsination eventhough you will wash it off, cells can get extra damage while you are doing your counting so your results can be affected by it. Make sure you seed enough amount of cell for your experiment, and check your cell confluency before starting to harvest them, I think it's enough. Also the time of trypsination is also an important factor, so try to not over extend it, and be carefully while washing you can wash off some of your cells which is not related of course to natural cell death so it can affect the correctness of your results. Make soft and gentle washings.
In case this could help you, before Annexin assay, I firstly collect supernatant to recover the dead cells and count them separately, and then count the live cells harvested by trypsinisation and washed with PBS, in order to quantify the total number of cells, dead and live you had before, as reference for the assay results. After this, I mix the two groups of cells and perform the assay. I agree Iskander recommendation in relation to time of trypsination and soft washings in order to care the cells.
As per the protocol, the number of cells you should count before staining with the dye, it is recommended by the manufacturer for proper cell staining.
If you are resuspending the pellet in the binding buffer after twice or thrice washing with PBS, you should count your cells in binding buffer with 0.4% of trypan blue with a hemocytometer, and count both the population dead and live.
In apoptosis study, live cells show only weak annexin V staining of the cellular membrane, while apoptotic cells show a significantly higher degree of surface labeling. Dead cells show both membranes (phospholipid phosphatidylserine (PS)) staining by annexin V and strong nuclear staining from the propidium iodide.
Dear Han,
I would suggest and second Harald Kühnel
method. After trypsinization, wash your cells once with PBS and then re-suspend in the 1mL PBS. Now take 10 uL of this cell suspension, mix it in 90 uL of PBS and count your cells. calculate per mL cells according to the formula.Then proceed for the second wash and discard supernatent, followed by the Annexin/PI staining protocol.
Hope this is helpful.
- Badges
- Science method