The cell membrane is the barrier for the exchange of substances between the cell and the external environment, and is a semi-permeable membrane that can selectively control the movement of substances in and out of the cell.
Principle
The basic principle of the cell membrane permeability measurement experiment is that if the red blood cells are placed in a hypotonic solution, due to the intracellular solute concentration is higher than the extracellular, so the liquid quickly enters the cell, so that the cell membrane is distended, hemoglobin escapes, that is, hemolysis occurs. If the erythrocytes are placed in a variety of isotonic solutions, the erythrocyte membrane of various solute molecules of different permeability, some solute molecules can be permeable, some can not be permeable; even if they can be permeable, the speed also varies. Therefore, when the solute molecules that are easily permeable enter the erythrocyte, the concentration of solute molecules increases with the intracellular solute molecules, leading to water intake, swelling of the erythrocyte, and the eventual rupture of the cell membrane resulting in hemolysis. At this point, light passes more easily through the solution, and the originally opaque erythrocyte suspension suddenly becomes a red transparent hemoglobin solution. Depending on the rate of solute permeation, the time to hemolysis varies. Therefore, the magnitude of cell membrane permeability to various substances can be estimated by measuring the hemolysis time.
Operation method
Cell membrane permeability assay experiment
Principle
The basic principle of the cell membrane permeability measurement experiment is that if the red blood cells are placed in a hypotonic solution, due to the intracellular solute concentration is higher than the extracellular, so the liquid quickly enters the cell, so that the cell membrane is distended, hemoglobin escapes, that is, hemolysis occurs. If the erythrocytes are placed in a variety of isotonic solutions, the erythrocyte membrane of various solute molecules of different permeability, some solute molecules can be permeable, some can not be permeable; even if they can be permeable, the speed also varies. Therefore, when the solute molecules that are easily permeable enter the erythrocyte, the concentration of solute molecules increases with the intracellular solute molecules, leading to water intake, swelling of the erythrocyte, and the eventual rupture of the cell membrane resulting in hemolysis. At this point, light passes more easily through the solution, and the originally opaque erythrocyte suspension suddenly becomes a red transparent hemoglobin solution. Depending on the rate of solute permeation, the time to hemolysis varies. Therefore, the magnitude of cell membrane permeability to various substances can be estimated by measuring the hemolysis time.
Materials and Instruments
Equipment: Move The basic procedure of the cell membrane permeability assay can be divided into the following steps: Caveat Test tubes should be numbered according to the type of solution to be contained in the experiment, and pipettes should be numbered accordingly, so as not to be confused, in order to ensure the accuracy of the results. For more product details, please visit Aladdin Scientific website.
Test tubes, test tube rack, marker, dropper, carrier sheet, cover sheet, forceps, filter paper, microscope.
Reagents:
① 10% sheep erythrocyte suspension (take 10 ml of mouse blood added to 90 ml of saline and mix)
② rabbit red blood cell suspension or mouse red blood cell suspension
③ Distilled water
③ Distilled water ④ 0.17 mol/L NaCl solution
⑤ 0.17 mol/L NH4Cl solution ⑥ 0.32 mol/L glucose solution
⑥ 0.32 mol/L glucose solution ⑦ 0.32 mol/L glycerol solution
⑦ 0.32 mol/L glycerol
⑧ 0.8 mol/L methanol solution
⑨ 0.8 mol/L glycerol solution
⑩ Chloroform
⑪ M-buffer (Imidazole, 3.404 g; KCl, 3.7 g; MgCl2▪6 H2O, 101.65 mg; ECTA, 380.35 mg; EDTA, 29.224 mg; mercaptoethanol, 0.07 ml; glycerol, 297 ml; and distilled water to 1000 ml. pH was adjusted to 7.2 with 1mol/L HC1 at room temperature. Adjust pH to 7.2 with 1 mol/L HC1 and store at room temperature.)
⑫ 2% Triton X-100 solution (Measure 2 ml of Triton X-100 (polyethylene glycol octyl phenyl ether) solution and add 98 ml of M-buffer to it.)
A Gently shake a test tube containing a suspension of sheep's blood erythrocytes.
B Observe the hemolysis of erythrocytes in hypotonic solution Add 0.3 ml of erythrocyte suspension to a test tube, add 3 ml of distilled water, and shake gently to mix well, and observe the change of the color of the solution. Observe the change in color of the solution. Observe the change in color of the solution. If the solution changes from an opaque red color (you cannot see the writing on the paper at the back of the test tube) to a clarified red color (you can see the writing on the paper clearly at this point), hemolysis has occurred. Record the time.
C Determining the Selective Permeability of Red Blood Cells to Various Substances Take 8 test tubes and add 0.17 mol/L sodium chloride solution, 0.17 mol/L ammonium chloride solution, 0.32 mol/L glucose solution, 0.32 mol/L glycerol, 0.8 mol/L methanol solution, 0.8 mol/L glycerin solution, chloroform, and 3 ml each of 2% Triton X- 100 solution. 3 ml each of chloroform, 2% Triton X-100 solution, after labeling, each tube was filled with 2 drops of human erythrocyte suspension, mixed well and left at room temperature.