Establishing a Müller Glial Cell Culture Obtained from Mouse Retinas
DEŞİFRE METNİ
Prepare papain DNase I dissociation mixture as described in the manuscript. Now, with an enlarged tipped transfer pipette, pick up the retinas. Wait until the retinas settle at the bottom of the tip, and release the retinas, without excessive HBSS, into the tube containing papain DNase I mixture.
Next, place the tube on a nutator in the incubator and incubate for 10 minutes at 37 degrees Celsius and with 5% carbon dioxide. Next, dissociate the cells by carefully pipetting up and down with a 1-milliliter pipette. After cells are dissociated, add 275 microliters of ovomucoid protease inhibitor from the papain dissociation kit to neutralize the papain, and mix gently by pipetting up and down.
Place tubes into a centrifuge, and spin at 4 degrees Celsius for 8 minutes at a relative centrifugal force of 300. Add epidermal growth factor to the calculated volume of growth medium prewarmed at 37 degrees Celsius. Remove the tubes carefully from the centrifuge.
Without touching the pellet at the bottom of the tube, remove the supernatant carefully and entirely. Now, resuspend the cell pellet with 500 microliters of epidermal growth factor-supplemented growth medium. Then, transfer the cell suspension into one well of the labeled 12-well plate. Rinse the tube with another 500 microliters of the epidermal growth factor-supplemented growth medium, and add it to the well.
Rock the well plate carefully. Then, place the plate into the incubator at 37 degrees Celsius with carbon dioxide. Begin by checking for 90% to 100% cell confluency. Next, remove the medium, and add 1 milliliter of cold HBSS to wash the well. Gently rock the plate and remove HBSS without any left traces.
Later, add 500 microliters of a prewarmed trypsin-containing solution to detach the cells from the well. Rock gently, and incubate for 2 minutes in 37 degrees Celsius incubator. After moving the plate from the incubator to the biosafety cabinet, aspirate the trypsin-containing solution while tilting. Disperse it carefully and slowly over the well several times, until the cells detach completely.
Next, transfer this cell suspension to a sterile 1.5-milliliter tube, and put tubes into the centrifuge. Spin at 300 times g for 8 minutes at 4 degrees Celsius, and bring tubes back into the biosafety cabinet. Remove the supernatant without touching the pellet. Now, carefully resuspend the cell pellet by adding 600 microliters of prewarmed growth medium and pipetting up and down approximately 30 to 40 times.
