MTT colorimetric method to detect the biological activity of IL-2

【Fundamental】
The IL-2 biological activity detection method is to detect the ability of IL-2 to promote the proliferation of target cells (or reactive cells). The level of IL-2 response cell proliferation can be used as an indicator system for DNA synthesis or enzyme activity in proliferating cells, by measuring 3H-TdRDNA incorporation or MTT colorimetric OD value, and indirectly measuring IL-2 organisms by comparison with standards Active unit. The following three types of cells can be used as IL-2 detection cells: â‘  IL-2 dependent cell lines, such as CTLL, CTLL-2, especially the latter are most commonly used; â‘¡ mitogen activated T lymphocytes; â‘¢ mouse thymus cells. The following mainly introduces the use of CTLL-2 cells as reactive cells to detect the biological activity of IL-2, and includes two basic methods of MTT colorimetry and 3H-TdR incorporation. For the principle, please refer to the biological activity detection section of IL-1.
【Materials and reagents】
(1) CTLL-2 cells: as reactive cells or target cells.
(2) Sample to be tested: multiple dilutions starting at 1: 2, at least 6 dilutions.
(3) IL-2 standard product: same as above, at least 6 different dilutions
(4) 10% FCS-RPMI-1640 complete medium
(5) MTT: MTT solution with a concentration of 5mg / ml is prepared with PBS, and sterilized and impurities are filtered with a 0.22μm filter membrane, and stored at 4 ° C in the dark.
(6) Acidified isopropanol: Add 0.4ml 36% HCl to 100ml isopropanol to become 0.04mol / L HCl isopropanol.
(7) 96-well cell culture plate, graduated pipette, capillary pipette, sampler (head), graduated centrifuge tube, centrifuge, cell counting plate, inverted microscope, ultra-clean workbench, CO2 incubator, enzyme label analyzer, 570nm and 630nm filters.
【Steps】
(1) Wash CTLL-2 cells twice with 10% FCS-RPMI-1640 complete culture medium, centrifuge at 1000r / min for 5min to remove the original growth medium (containing IL-2);
(2) Adjust the cell concentration to 1 × 105 / ml with 10% FCF-RPMI-RPMI-1640 medium;
(3) Add IL-2 standards and samples to be tested at different dilution ratios to 96-well culture plates, 100 μl / well, each with 3 replicate wells, and set culture medium controls at the same time;
(4) Add CTLL-2 cell suspension to each well, 100μl / well;
(5) Incubate at 37 ° C and 5% CO2 incubator for 24h (18-24h is acceptable);
(6) When the cells are cultured to 18-24h, add MTT solution to each well and continue to culture for 10h at 10μl / well;
(7) Take out the culture plate and gently aspirate 100μl of supernatant from each well and discard it. Then add enzymatic isopropanol or DMSO, 100μl / well, set at room temperature for 10-20min, vortex and shake, mix well to make the MTT-formaldehyde product fully dissolved;
(8) Use a microplate reader to detect the wavelength of 570nm and the reference wavelength of 630nm, and measure the OD of each well. The measurement should be completed within 1h after the addition of acidified isopropanol.
【Result analysis】
(1) The OD value of each well should be taken as the average of 3 multiple wells. The final OD value of each well should be OD570nm-OD630nm, and then subtract the OD value of the control well of the culture solution;
(2) Calculate the IL-2 activity unit according to the probabilistic unit analysis method (see the IL-1 biological activity detection method section): the sample IL-2 activity unit is calculated using the following formula:

Sample IL-2 activity unit (U / ml)
=
Sample dilution up to 50% of the standard's maximum OD value
×
Standard product IL-2 activity unit
(U / ml)
Standard dilution corresponding to 50% of maximum OD value
【Precautions】
(1) The survival rate of CTLL-2 cells should be> 95% (the cells have good refractive index and full shape), and the operation should not be too violent during washing, because the CTLL-2 cell membrane is extremely brittle and easily broken, which affects the test results.
(2) CTLL-2 cells should be washed thoroughly, because the original growth medium contains IL-2.
(3) CTLL-2 cells also have a proliferation response to mouse IL-4. If the sample contains mouse IL-4, it will affect the accuracy of the test results. At this time, the anti-mouse IL-4 McAb can be used to process the sample to be tested . However, CTLL-2 cells had no proliferation response to human IL-4.
(4) The optimal final concentration of CTLL-2 should be 1 × 104 / well, and should be evenly distributed.
(5) CTLL-2 cells are difficult to recover after cryopreservation, and long-term culture can easily produce mutations that interfere with IL-2 activity measurement, so care should be taken.
(6) The IL-2 standard and the sample to be tested must have at least 6 dilution ratios starting from 1: 2. The samples should be added sequentially from low concentration to high concentration, and the sampler head cannot be shared.
(7) The best time to add MTT should be when the cells in the culture medium control (without IL-2) all die, the general time is when the cells are cultured to 18-24h.
(8) After adding enzymatic isopropanol, the OD should be measured within 1h. If it cannot be measured within 1 hour, the 96-well culture plate without acidified isopropanol can be temporarily stored in the refrigerator at 4 ℃. Before the measurement, take it out at room temperature for several minutes, and then add enzymatic isopropanol to measure the OD value.
(9) The phenol red contained in the RPMI-1640 culture liquid can interfere with the OD value determination, and the addition of enzymatic isopropanol can change the phenol red in the culture liquid from red to yellow under acidic conditions, which can Eliminate the interference of red on the determination of OD value; in addition, the establishment of culture medium control can further exclude the influence of culture medium on the determination of OD value.
(10) There are many calculation methods for IL-2 activity units. In addition to the methods described in the IL-1 activity unit calculation method, IL-2 activity units can also be calculated by the normal probability paper method and the probability unit method. The results can be processed by computer to obtain the IL-2 activity unit. For the calculation method, please refer to the relevant content of medical statistics.

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