Hi Ayat AL-Laaeiby,

If the engulfed cells are bacteria and you want to plate the lysate to find out the CFU count, then I would suggest you to use 0.02% SDS in 1X PBS for lysis of the macrophage. This much lower concentration of SDS will not do lysis of the engulfed bacteria. After adding the lysis buffer gently mix it by pipetting ( Don't Vortex) and plate it within 5 minutes.

Best,

Biswaranjan Pradhan

Ph.D. Student, SBS,

NISER Bhubaneswar

Hi Ayate

This method me be helpfull

Cell Lysis

1.         There are a lot of ways to do this and it depends mainly on what you are going to try to look for.  If you are doing a standard western blot for a cytoplasmic or cell surface protein (meaning basically non-nuclear) we will do a lysis in NET with 0.5% Triton X100

                        NET:   20 mM Tris

                                    100 mM NaCl

                                    1 mM EDTA

            Make a 10% Triton X100 stock solution in water and dilute to 0.5% in NET to make lysis buffer.

            You also need to add protease and maybe phosphatase inhibitors. 

Protease inhibitors are in a cocktail from BD-Pharmingen that is 50x in ethanol and is stored in the ‑20oC.

Phosphatase inhibitors are in two cocktails, both in the refrigerator.  Add each at 50x.

After making lysis buffer, keep everything on ice at all times!!!

2.         Spin cells to be lysed down in 15 or 50 mL centrifuge tubes.  Aspirate off media, leaving around 1 mL.  Transfer that 1 mL to a microcentrifuge tube and pulse spin the cells to pellet (spin for 5-10 seconds at max speed in the microcentrifuge).

3.         Resuspend cells in lysis buffer.  Usually for cell lines add around 20 mL of lysis buffer per million cells.  For primary lymphocytes, add 10 mL lysis buffer per million cells.

4.         Let cells sit in lysis buffer on ice for 10-15 minutes to ensure complete lysis.

5.         While cells are lysing on ice, make sure the refrigerated microcentrifuge is set to 2-4oC.

6.         Spin cells at max speed for 10 minutes at 2-4oC to pellet nuclei.

7.         Harvest supernatants into fresh tubes.  At this point you can

a.         do a BCA protein assay, dilute lysates to appropriate concentrations with SDS-loading buffer, and boil for ten minutes, then freeze at -20oC or

b.         freeze lysates and quantitate proteins later

Thank you very much for your answers 

Hi Ayat,

Because macrophages are adherent, after co-incubation, wash your plate with cold PBS to remove any non-ingested cells and then lyse in the normal way as you would for a Western Blot.

Thank you very much Carol for your answer.