The overall aim of this procedure is to isolate and immunostain lymphocytes and dendritic cells from neuron payers patches, using two parallel experimental approaches. First, excise fresh payers patches from the mouse, small intestine. Then physically dissociate the payer's patches, harvest the tissue and pass it through a mesh filter to generate a single cell suspension for flow cytometry analysis.
Harvest the cells by centrifugation and distribute aliquots in a 96 well round bottom plate. Next, label the cells with antibodies against cell type specific surface markers. Immediately proceed to flow cytometric analysis to enumerate the specific cell types In parallel isolate payer's patches and snap freeze tissue samples.
In OCT blocks, cryo section, the samples label with antibodies against cell type specific surface markers and then visualized by confocal scanning laser microscopy taken together. These methods will quantify B cells, T cells, and dendritic cells in specific compartments within the payer's patch tissues. Generally, individuals new to this method will struggle because PI's patch tissues are extremely fragile and can be easily damaged.
During handling. Lay the freshly isolated small intestine on a bed of moist paper towels or Kim wipes. Then wet the small intestine gently with saline to prevent tissue dehydration.
Now, visually identify individual payers patches located on the anti mesenteric side of the intestine. Typically, a single mouse has five to 10 visible payers patches that are evenly distributed from the duodenum to ileum. Using curved surgical scissors generally excise the individual payer's patches.
Place them in cold hank's balance sort solution, maintaining the tissue at four degrees Celsius to preserve cell viability. Next, transfer the payer's patches into five milliliters of spleen dissociation medium and shake at 250 RPM for 15 to 20 minutes at 37 degrees Celsius. Then to generate a single cell suspension, place the sample on a sterile 70 micron nylon mesh cell strainer and forcibly grind the tissue into the mesh using the base of a plunger from a one cc syringe to remove remaining cellular aggregates.
Decant the cell suspension through a second 70 micron cell, strainer and collect the cells in a 15 or 50 milliliter conical tube. Dispense 10 to the five cells per well into a 96 well round bottom plate pellet cells by centrifugation for five minutes at 1000 times. G.Then decant the supernatant by gently inverting the plate.
Now resuspend the cells in FC block buffer and incubate on ice for 15 minutes. Next, add Fluor conjugated antibodies directly to cell suspensions at the desired dilution and incubate on ice wash cells with 100 microliters of flow buffer. Then resuspend the cells in 400 microliters of fixation buffer to analyze the cells by flow cytometry to isolate payers patches for cryosectioning.
Cut 0.5 centimeter transverse segments of small intestine containing a single payer's patch. Transfer the tissue to a Petri dish containing PBS. Then place the intestinal tissue segments vertically inside base molds containing OCT.
Immerse the molds in liquid nitrogen and allow tissue to freeze completely. Remove the frozen base molds from the liquid nitrogen using forceps. Allow the mold to warm up room temperature just long enough for the edges of the OCT block to soften.
Then remove the frozen block of OCT from the mold. Invert the mold and press gently on the back to eject the block onto a clean four by four centimeter piece of aluminum foil. Immediately wrap the tissue in the foil and place in a labeled specimen bag stored in liquid nitrogen or on dry ice cryo section the tissue using a LI a CM 3 0 5 0 s cryo microtome or equivalent.
Set the chamber temperature on the cryostat to minus 21 degrees Celsius. Ideally cut sections of 12 to 14 microns in thickness. Collect the sections onto Supercross plus glass microscope slides.
Inspect the samples visually using a standard low power light microscope. Immerse the slides in acetone for two minutes. Be mindful that prolonged incubation may cause tissues to detach from the slide.
Wash the samples three times with PBST. Then encircle the sections with an image hydrophobic pen. Now incubate the samples in block buffer, overlay the tissue sections with primary antibody solution.
Dry the area around the sections using Kim wipes or other absorbent tissue without touching the actual tissue sections. Using a pipette or dropper, gently place a drop of prolonged gold mounting medium directly onto the section. Apply a glass cover slip, avoiding air bubbles and blotting any excess mounting.
Medium fax analysis of mono dispersed suspensions of total payers patch cells reveals a clear distinction between good and poor cell preparations in good cell preparations. With over 80%viability, the vast majority of cells demonstrate high forward scatter indicating high cell volume and low side scatter consistent with low cell granularity. In this experiment, poor cell preparation was deliberately prepared to demonstrate that in a bad population, the majority of cells exhibit low FSC, high SSC, and are outside the indicated gait R one.
These cells are likely undergoing apoptosis and or necrosis. Here a cocktail of up to four different Fluor. Four conjugated monoclonal antibodies to discriminate among a variety of cell types.
In payers patch single cell suspensions were used labeling with a cluster of differentiation markers. CD 19 and B two 20 reveal that B cells constitute around 60%of the gated payers. Patch cell population specific T-cell subsets can be easily enumerated by double labeling with antibodies against CD three and CD four, or CD three and CD eight, CD four positive and CD eight positive T cells constitute approximately 23%and 9%respectively of the total payers patch cells.
DC's subsets, they would CD 11 C positive and CD 1 0 3 positive can also be enumerated by this method. CD 11 C positive dcs contribute to about 8%of the total gated population, which is roughly equivalent to 10, 000 dcs per payers patch. Among the population of CD 11 C positive cells, approximately 4.7%were double positive for B two 20 positive.
Similar results are obtained when payers patched cells are collected from C 57 black six mice. Poor cell preparations can lead to highly misleading results, largely because dead or dying cells tend to bind antibodies non-specifically. Typically, these lead to an overrepresentation of B 2 2 0 positive and CD three positive double positive cells as well as B two 20 positive B cells and CD four positive T cells double positive cells.
The mouse payers patch cryo sections are amenable to histopathologic analysis as well as immuno labeling. While the power of in sections provide more resolution at the cellular level when stained by h and e, the light and dark zones of payers patch germinal centers are more easily demarcated in cryo section sections. In this typical payers patch cryo section, anti CD three antibodies, label the T cells anti CD 11 C antibodies.
Highlight dendritic cells and anti B two 20 antibodies. Highlight the B cells After its development. This technique paved the way for researchers in the field to understand how specific cell types like dendritic cells within gut-associated lymphoid tissues respond to intestinal antigens and microbial pathogens.
This information has influenced mucosal vaccine design and development.
