The objective:
To compare the antimicrobial peptides compositions in neutrophils with stimulation and without stimulation.
Salah A. Alshehade
Collect a blood sample from a healthy individual using a sterile syringe and needle. The volume of the blood sample will depend on the specific experiment and the number of neutrophils required. Transfer the blood sample to a sterile centrifuge tube containing an anticoagulant, such as ethylenediaminetetraacetic acid (EDTA) or heparin, to prevent clotting. Gently mix the blood and anticoagulant by inverting the tube several times. Centrifuge the blood sample at a low speed (e.g., 200-300 x g) for 10 minutes to separate the blood components. This step will result in the formation of three layers: a top layer of plasma, a middle layer of white blood cells (including neutrophils), and a bottom layer of red blood cells. Carefully collect the middle layer of white blood cells using a pipette and transfer them to a new sterile centrifuge tube. Add a red blood cell lysis buffer (e.g., ammonium chloride-potassium (ACK) lysing buffer) to the tube containing the white blood cells to remove the red blood cells. Incubate the tube for 5-10 minutes at room temperature. Centrifuge the tube at a low speed (e.g., 200-300 x g) for 10 minutes to pellet the white blood cells. Discard the supernatant and resuspend the white blood cell pellet in a suitable buffer, such as phosphate-buffered saline (PBS) or Hank's balanced salt solution (HBSS). Count the number of neutrophils using a hemocytometer or an automated cell counter. Adjust the cell concentration as needed for your experiment. Prepare a stimulation buffer by adding a suitable concentration of a stimulant, such as phorbol 12-myristate 13-acetate (PMA) or N-formylmethionyl-leucyl-phenylalanine (fMLP), to a buffer solution (e.g., PBS or HBSS). Transfer the desired number of neutrophils (from the previous step) to separate sterile tubes. Label one tube as the "stimulated" sample and the other as the "unstimulated" sample. Add the stimulation buffer to the "stimulated" sample, while adding an equal volume of the buffer solution (without the stimulant) to the "unstimulated" sample. Incubate both samples for a specific time period (e.g., 30 minutes) at 37°C to allow degranulation to occur in the stimulated sample. After the incubation period, centrifuge both samples at a low speed (e.g., 200-300 x g) for 10 minutes to pellet the neutrophils. Collect the supernatant from each tube, which will contain the released antimicrobial peptides from the degranulated neutrophils. Store the supernatant samples at -80°C or proceed with the analysis of the antimicrobial peptides using suitable techniques, such as radial diffusion assays or mass spectrometry, to compare the compositions of the peptides in the stimulated and unstimulated samples
Polymorphonuclear leukocytes (PMNs or neutrophils) extraction from blood:
Degranulation of PMNs:
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