Preparation of HeLa Cells for Flow Cytometry

Reagents: 
Release Buffer:Phosphate-buffered Saline, pH 7.4
                          10mM EDTA

FACS Buffer:   Phosphate-buffered saline, pH 7.4
                       5% FBS (Fetal Bovine Serum)
                       0.02% sodium azide
                       Ice Cold.
If you are going to fix cells:
                       Phosphate buffered saline        
              2% formaldehyde (w/v)  (from stock 37%)

Protocol:

(This procedure is for HeLa cell line which is adherent cell line, they need to be detached from the cell culture plate to proceed with flow cytometry experiment protocol. For non-adherent cell lines, spin out the number of cells and proceed with the step number 4.  And To have the sufficient number of cells (~500,000), its a good idea to start with 1-2 million cells, as you might loose some cells during trypsinizing, washing and staining procedures).


Releasing the adherent cell line HeLa cells from the culture plate.

            Before proceeding to release the cells from plate, make sure that cells are around 50- 60% confluent. If confluence is good, then these cells are also great for preparing controls and staining. HeLa cells can be released from plate using EDTA. and this EDTA can be washed off with the help of PBS.
        * Use the pippete and take out all the media present in the plate.
        * Add 3mL of PBS/10mM EDTA to each plate and incubate for 5-10 minutes.
           (If you want speed up, then you can keep incubate in 37°C incubator for 2-3 minutes.
      * look under microscope to be sure that cells have detached from the surface. cells will look rounded and loose.. 

        * add 4mL of media with FBS. 

        * Pippette up and down for several times to detach all the cells which have not detached.
        * Transfer them 15mL tubes and Spin them down.
        * Take out the supernatant and do not disturb the pellet.
       * Add 3mL of PBS to each tube and spin them. (This is a washing step). discard pellet and repeat it one more time.
        * Add 1mL of PBS buffer to each tube and resuspend the pellet. (Now single cell suspension is ready and good to proceed to record the data in a flow cytometer).
        * Transfer these suspension to 5mL FACS Tubes. (Make Sure to label them properly).

(If you have transfected the cells, you can stop at this step and Run the sample tubes in flow cytometer).

Staining with Primary Antibody:

       For this protocol, you must think about the controls required for the experiment. For simple single staining, you should have unstained cells as negative or irrelevant antibody to set the baseline before running the samples.
       *Spin out equal number of cells for staining and preparing controls.
       *Resuspend the pellet in 50µL of diluted antibody (Titrated Antibody Concentration).
       *Incubate the tubes on ice for 30-60 minutes (or as per the manufacture's suggestion).
       *Add 500µL of FACS buffer to each tube, resuspend and spin them and discard the supernatant. Repeat this step 2 more times with FACS buffer.

Staining with Secondary Antibody:



      After staining with primary antibody, Spin down the cells.
      *Resuspend the pellet in 50µL of diluted secondary antibody (Titrated Antibody Concentration).
      *Incubate the tubes on ice for 30 minutes. (or as per the manufacture's suggestion)
      * Add 500µL of FACS buffer to each tube and spin the tubes. Discard the supernatant. repeat this step one more time with FACS buffer.

Staining with Propidium Iodide (PI):
            Propidium Iodide (PI) is a DNA-binding dye.. It binds only to DNA (to RNA also). This dye can be used to study the cell cycle and to discriminate the dead the cells from the prepared cells for flow cytometry. PI can not penetrate through the cell wall and bind to DNA. It will bind to the DNA of the dead cells as their membrane is compromised. So, this can be helpful in excluding dead cells from live cells during flow cytometry analysis.