2.3. Flow cytometric Analysis of MAPKs mediated signaling molecules

Patients in both groups were assessed for the expression levels of MAPKs signaling components pre and post PEMFT and LLLT in the peripheral Lymphocytes. Two blood samples (5 cc of venous blood using EDTA as the anticoagulant) were taken, in each time of PEMFT and LLLT applications, from every included patient, one before and the other after the applications, the pre-application sampling were taken to assess if there were cumulative effect of the PEMFT or LLLT from the previous application or not to be taken into consideration during calculation of the results. Peripheral mononuclear cell suspension pellets were obtained from each anti-coagulated blood sample using density gradient centrifugation on Ficoll-Hypaque media, were the blood samples layered onto a Ficoll-sodium metrizoate gradient of specific density following centrifugation, mononuclear cell pellets were collected from the plasma-Ficoll interface.

Culture media was prepared by transferring 9 ml of DMEM media (lot n s13365L0103 biowest) to the labeled flask, 100 µl Fungizone (10 µl/1 ml media) and 100 µl Pen/Strep solution (10 µl/1 ml media) were pipette to the same flask. 1 ml of suspended cell pellet was added to the previous mixture with gentle mixing. The mixture was incubated for 2 h at 37 0 C in 5% CO₂. The flask was removed from the incubator and placed in the hood. The media containing non-adherent cells was collected from the flask by a sterile pipette.

After isolation of lymphocytes, cells were suspended at 1×106/ml in tissue culture medium (αMEM) plus 10% fetal bovine serum (FBS lot n 41G5834K)) at 37° water bath. Activation was done for 10 min using 40 nM phorbol myristate acetate [(PMA; #P8139), which obtained from Sigma, and used at a 40 mM working dilution dissolved in 100% ethanol and the concentration adjusted to 1×10⁶/ml in α MEM (lot number M0200) (modified Egle media) containing 10% fetal calf serum]. While maintaining sample at 37°, fixation was done (at 37° for 10 min) by adding 10% formaldehyde (Ultrapure M Grade) to give a final concentration of 2% formaldehyde. Tubes were placed on ice for 2–3 min, then 100% ice cold methanol was added followed by gentle vortex mixing, to give a final concentration of 90% methanol, held on ice for 30 min, then processed for antibody labeling. The tubes were centrifuged, aspirated, and washed once using 2 ml PBS containing 4% FBS. Centrifugation and re-suspended at 10⁶ cells in 100 ml PBS plus 4% FBS was done. Then Labeling with phospho-specific polyclonal antibody (from Abcam Company) to ERK1/2(lot n s13365L0103), P38 (lot n ab170099) and JNK (lot n ab31419) separately was done for 15 min at room temperature, then washed once using 2 ml PBS plus 4% FBS, all these antibodies used at a 1/100 dilution. The samples were run on flow cytometer (BD FACS Calibur flow cytometer, USA); fluorescence signals were collected using band pass filters excitation at 525 nm and emission at 575 nm. The expression levels were automatically calculated by the flow cytometry by measuring the percent (%) of expression in the activated lymphocytes appeared at the upper right quadrant of the graph.