Das

The overall goal of this procedure is to measure the effects of pathogenic challenge on small intestine organoids with an emphasis on normalization techniques against the total cell number.This method could help answer key questions in the innate as well as mucosal immunology fields by providing a means to investigate host pathogen interactions of bacteria with intestinal epithelial cells in vitro.Essential to this procedure is the method for normalization against the total cell number, a necessity when measuring organoid secreted proteins via techniques such as ELISA.To harvest mouse small intestinal crypts for organoid culture, begin by using a sterile glass slide to gently scrape away the villi from the luminal surface of the small intestine.Next, use dissecting scissors to cut the tissue into 1-2 centimeter length strips.And place the strips into a 50 milliliter conical tube containing 10 milliliters of ice cold PBS.Mix the contents by gentle inversion and allow the tissue contents to settle to the bottom of the tube.Then, aspirate the PBS and wash the strips three more times in 10 milliliters of fresh PBS, each time in the same manner.At the end of the final wash, place the tube on ice for ten minutes.Then, replace the PBS with 25 milliliters of PBS supplemented with 2 millimolar EDTA, in an ice bucket on a rocking platform for 45 minutes.At the end of the incubation, allow the tissue strips to settle to the bottom of the tube and replace the PBS-EDTA with 10 milliliters of PBS supplemented with 10%FBS.Shake the tube vigorously by hand ten times.Then, allow the tissue to settle to the bottom of the tube and transfer the supernatant to a 15 milliliter conical tube labeled, Fraction 1.Agitate the tissues in PBS FBS five more times.Transferring the supernatant into a new fraction tube each time.After the last agitation, centrifuge all of the samples and resuspend the pellets in five milliliters of prewarmed DMEM/F12 with no added growth factors.Now, spin down the samples again and aspirate four milliliters of the supernatant from each tube.Resuspend the pellets in the last milliliter of remaining medium and use 20 microliter aliquots from each fraction to visualize the crypts and the debris on glass slides.After pooling the appropriate fractions to achieve the greatest percentage of crypt to debris ratio, centrifuge the pooled fractions and aspirate all but the last 50-100 microliters of the supernatants.Keeping the tubes on ice, add 1 milliliter of protein matrix to the pooled fractions, pipetting up and down slowly to prevent the addition of air bubbles.Then, add 50 microliters of the protein matrix/crypt suspension into the middle of each well of 37