Isolation of Uterine Innate Lymphoid Cells for Analysis by Flow Cytometry

This is a method to isolate uterine lymphoid cells from both pregnant and non-pregnant mice. This method can be used for multiple downstream applications such as FACS phenotyping, cell sorting, functional assays, RNA-seq, and proteomics. The protocol here demonstrates how to phenotype group 1 uterine innate lymphoid cells by flow cytometry.

Our method enables assessment of the composition and functional characteristics of different lymphocytes subpopulations present in the uterine tissue of pregnant and non-pregnant animals. This protocol allows for isolation of uterine lymphocytes while preserving surface expression of protein, cell viability, and functionality. Therefore it is suitable for a range of subsequent applications.

Fetus removal at the beginning of the experiment and leukocyte collection are technically challenging steps. Since it is a lengthy experiment, particular attention and focus are needed once you've reached the antibody staining steps. To begin, transfer the uterus tissue harvested from a pregnant C57 black six female mouse into a sterile petri dish, then using sterile instruments gently remove the fat surrounding the tissue, taking care not to let the tissue dry.

Remove the fetuses by dissecting each implantation site with sterile instruments, then return the uterus to its original five milliliter collection tube containing one milliliter of collection media. Using scissors, mince the tissue in the collection tube, then place the tube in a 37 degrees Celsius water bath. Next add three milliliters of pre warmed enzymatic digestion mix to the tube.

Incubate the tube for 30 minutes at 37 degrees Celsius with agitation to enhance the enzymatic digestion activity. After the incubation is complete, vortex the tube, and keep it on ice to inhibit the enzymatic activity, then transfer the digestive tissue into a properly labeled 15 milliliter centrifuge tube. Rinse the five milliliter collection tube with a total of 10 milliliters of ice cold five millimolar EDTA PBS solution to collect all the remaining tissue and transfer it into the 15 milliliter centrifuge tube.

Centrifuge the digested tissue sample for 10 minutes at 400 times G.Discard the supernatant, gently flick the pellet, and then resuspended in 10 milliliters of pre-warmed five millimolar EDTA PBS solution. To reduce cell clumping, incubate the sample at 37 degrees Celsius with agitation, followed by vortexing on high for 10 seconds. For removing cell clumps in undeassociated tissue, place a 70 micrometer strainer on top of a sterile 50 milliliter centrifuge tube, then using the plunger of a sterile one milliliter syringe, force the digested tissue through the strainer into the tube.

Wash the strainer several times with a total of 10 milliliters of cold PBS to collect all the cells, then spend the 50 milliliter centrifuge tube for 10 minutes at 400 times G.After discarding the supernatant, the innate lymphoid cells may be isolated using either option A or option B.For option A, resuspend the pellet in five milliliters of 80%isotonic per call using a pipette voy. Then using the pipette voy on slow speed, carefully overlay eight milliliters, 40%per call solution, onto the resuspended pellet in a 15 milliliter centrifuge tube. Pipette slowly and continuously, holding the 15 milliliter to about a 45 degree angle.

Without disturbing the overlay, centrifuge the tube for 20 minutes at 850 times G with medium acceleration and minimum breaks at room temperature. Once the centrifugation is complete, carefully remove the tube from the centrifuge without disturbing the per call layers. Next without disturbing the ring of leukocytes at the interface of the two per call solutions, use a sterile pasteur pipette to discard all except 0.5 to one milliliter of the top per call layer.

While trying to keep the amount of per call solution sucked into the pipette to a minimum, carefully collect the ring of leukocytes, and transfer the cells into a new labeled 15 milliliter centrifuge tube. Add 10 milliliters of sterile RPMI medium supplemented with 10%heat and activated FBS to the cells, then centrifuge the cells for five minutes at 500 times G and four degrees Celsius. Discard the supernatant and proceed to red blood cell lysis.

To isolate the cells using option B, after passing the digested tissue through the cell strainer and centrifuging the result in cell suspension as demonstrated previously, resuspend the pellet with eight milliliters of 35%isotonic per call and RPMI medium, and transfer the cells into a 15 milliliter tube. Centrifuge the tube at 940 times G for 10 minutes at room temperature with medium acceleration and minimum breaks. Then using an aspirator or pipette voy, aspirate the supernatant carefully before resuspending the pellet in 14 milliliters of RPMI medium supplemented with 10%heat and activated FBS.

Centrifuge the sample again for five minutes at 500 times G and four degrees Celsius, then discard the supernatant by aspiration and proceed to red blood cell lysis. To lyse the red blood cells, resuspend the sample in three milliliters of one X red blood cell lysing solution and incubate for three minutes at room temperature, then add 10 milliliters of PBS into the sample to stop the reaction. Centrifuge the tube at 400 times G for five minutes after discarding the supernatant, add another 10 milliliters of PBS and repeat centrofugation.

Resuspend the pellet in one milliliter of RPMI medium supplemented with 10%heat inactivated FBS, then pass the in cell suspension through a sterile 70 micrometers cell strainer. After counting the cells adjust the concentration of the cell suspension to one to 2 million cells in 100 microliters of PBS and proceed to staining and facs analysis. Uterine group one ILCs include conventional NK cells, tissue resident NK cells, and group one ILCs with their percentages varying during life and pregnancy.

These subsets can be discriminated using flow cytometry by first gating cells on their ability to scatter light, then isolating single buyable CD 45 positive CD three negative CD 19 negative cells, and then identifying group one ILCs that are NK 1.1 and NKP 46 positive. Within group one ILCs, CD49A negative EMs positive are conventional NK cells. CD49A positive Ems positive cells are tissue resident NK cells.

And CD49 positive Ems negative cells are group one uterine ILCs. Staining of splenic and uterine lymphocytes with anti NK P46 and anti NK 1.1 antibodies shows that the splenic lymphocytes express higher amounts of NKP 46 on their surface than their uterine counterpart. In enzymatic tissue dissociation, surface epitopes can be altered depending on the enzymes used in the digestion medium.

For example, staining for the MHC CD49 NKG2A receptor is poor when liberase TM is used. However digestion with liberase DH preserves NKG2A recognition by the 1611 antibody clone. Around 6.5%of group one ILCs present in uterine tissue samples at gestation day 8.5 are blood derived.

Blood contaminants can be excluded through intravital staining with anti CD45 antibodies conjugated with a fluorochrome. When setting up for time meetings, you must take into consideration that mice are nocturnal. Therefore they should be set up as late as possible in the afternoon as mating will occur during the night.

Also when selecting females, you must ensure that they do not have a vaginal septum. We typically do flow cytometry analysis to look at many proteins on the outside of the cells and within the cells. We also do nucleic acid extraction to study gene expression, including RNA sequencing.

And we also do manage to around functional assays to study the responses of uterine innate lymphoid cells.