ELISA (Enzyme-Linked Immunosorbent Assay)
ELISA is a technique to detect and quantify substances like proteins, antibodies, and hormones. The target antigen is immobilized to the bottom of the well and then an antigen-specific detection antibody is added. The detection antibody makes it possible to measure results. The three most common formats of ELISA would be direct assay, indirect assay, and sandwich assay. In direct assay, the antigen immobilizes to the bottom of the well where a labeled antibody attaches to it. In indirect assay, the the antigen immobilizes to the bottom of the well and then a primary antibody binds to the antigen. After that, the labeled secondary antibody binds to the primary antibody. In sandwich assay, a pre-coated …show more content…
Before putting it in the biosafety cabinet, spray the bottles and gloves with 70% ethanol. Then, suck out the substrate and dispense in the organic waste flask. Wash all the flasks with PBS to remove any residue of the substrate and then dispense the liquid. After that, add trypsin/EDTA to remove the cells from the bottom of the bottle. Incubate the bottles for 5 minutes. Swirl the bottle around to make sure that trypsin removes all the cells from the bottle. Then, use a pipette to remove the substrate from the bottles and into centrifuge tubes. Then centrifuge the tubes for 1 minute at 1000xg. Make sure that the centrifuge tubes are properly balanced. If the centrifuge tubes do not have pellets at the bottom centrifuge again. Once back in the biosafety cabinet, remove the supernatant at the top of the centrifuge tubes until only the pellet remains. Discard the supernatant into the organic waste flask. Then in a well add trypan blue. Add the pellets into the well with trypan blue. Pipette up and down to make sure that the mixture is homogenous. The blue dye helps identify which cells are dead and which cells are alive. If the cell becomes blue, that means that it is dead. To view the cells, place a pipette of cells mixed with trypan blue at the edge of the hemocytometer and dispense the solution. The slide should already be on the hemocytometer before dispensing the liquid. Next, place the hemocytometer under a microscope …show more content…
The RPMI basal media and gentamycin need to be stored at 2-8C and the pen-strep, L-Glutamine, and FBS need to be stored at -20C. Before starting, remove the reagents from the freezer and place in the refrigerator to thaw overnight. Place the L-Glutamine in a water bath for 5 minutes at 37°C before starting. Shake the tube in order to ensure that all particles have dissolved. Before placing anything in the biosafety cabinet, spray the BSC with 70% ethanol as well as any material put inside the BSC. Use a sterile 500 mL bottle and label it with the media name, contents, expiry date, preparation date, and preparer’s name. Use a pipette to add 450 mL of RPMI basal media into the sterile bottle. Then add 50 mL of FBS into the sterile bottle and stir for two minutes to allow the FBS to be thoroughly mixed. Next, add 5 mL of 1X Pen-Strep and 5 mL of L-Glutamine. Finally, add 1 mL of 10 mg/mL of Gentamycin to obtain a concentration of 0.02 mg/mL. Stir the media once again. The RPMI media can be stored at 2-8°C for a month before
ELISA is a technique to detect and quantify substances like proteins, antibodies, and hormones. The target antigen is immobilized to the bottom of the well and then an antigen-specific detection antibody is added. The detection antibody makes it possible to measure results. The three most common formats of ELISA would be direct assay, indirect assay, and sandwich assay. In direct assay, the antigen immobilizes to the bottom of the well where a labeled antibody attaches to it. In indirect assay, the the antigen immobilizes to the bottom of the well and then a primary antibody binds to the antigen. After that, the labeled secondary antibody binds to the primary antibody. In sandwich assay, a pre-coated …show more content…
Before putting it in the biosafety cabinet, spray the bottles and gloves with 70% ethanol. Then, suck out the substrate and dispense in the organic waste flask. Wash all the flasks with PBS to remove any residue of the substrate and then dispense the liquid. After that, add trypsin/EDTA to remove the cells from the bottom of the bottle. Incubate the bottles for 5 minutes. Swirl the bottle around to make sure that trypsin removes all the cells from the bottle. Then, use a pipette to remove the substrate from the bottles and into centrifuge tubes. Then centrifuge the tubes for 1 minute at 1000xg. Make sure that the centrifuge tubes are properly balanced. If the centrifuge tubes do not have pellets at the bottom centrifuge again. Once back in the biosafety cabinet, remove the supernatant at the top of the centrifuge tubes until only the pellet remains. Discard the supernatant into the organic waste flask. Then in a well add trypan blue. Add the pellets into the well with trypan blue. Pipette up and down to make sure that the mixture is homogenous. The blue dye helps identify which cells are dead and which cells are alive. If the cell becomes blue, that means that it is dead. To view the cells, place a pipette of cells mixed with trypan blue at the edge of the hemocytometer and dispense the solution. The slide should already be on the hemocytometer before dispensing the liquid. Next, place the hemocytometer under a microscope …show more content…
The RPMI basal media and gentamycin need to be stored at 2-8C and the pen-strep, L-Glutamine, and FBS need to be stored at -20C. Before starting, remove the reagents from the freezer and place in the refrigerator to thaw overnight. Place the L-Glutamine in a water bath for 5 minutes at 37°C before starting. Shake the tube in order to ensure that all particles have dissolved. Before placing anything in the biosafety cabinet, spray the BSC with 70% ethanol as well as any material put inside the BSC. Use a sterile 500 mL bottle and label it with the media name, contents, expiry date, preparation date, and preparer’s name. Use a pipette to add 450 mL of RPMI basal media into the sterile bottle. Then add 50 mL of FBS into the sterile bottle and stir for two minutes to allow the FBS to be thoroughly mixed. Next, add 5 mL of 1X Pen-Strep and 5 mL of L-Glutamine. Finally, add 1 mL of 10 mg/mL of Gentamycin to obtain a concentration of 0.02 mg/mL. Stir the media once again. The RPMI media can be stored at 2-8°C for a month before