May 23, 2022
Intestinal Lamina Propria and Spleen Immune Cell Isolation
- rabdelha 1
- 1California Institute of Technology
Protocol Citation: rabdelha 2022. Intestinal Lamina Propria and Spleen Immune Cell Isolation . protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpbxo5lzp/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: May 12, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 62485
Keywords: Immune Cells Isolation, Intestinal Lamina Propria/Spleen, Flow Cytometry, ASAPCRN, intestinal lamina propria, spleen immune cell isolation, protocol details isolation of immune cell, immune cell, spleen, cell, cytometry, protocol details isolation
Abstract
This protocol details isolation of immune cells from intestinal lamina propria/spleen and flow cytometry.
Attachments
436-924.docx
105KB
Troubleshooting
Isolation of Immune Cells from Intestinal Lamina Propria/Spleen
Dissect the small and large intestines for isolation of intestinal lamina propria cells, place the small and large intestines immediately On ice cold PBS.
Open the intestines longitudinally after removing the mesenteric fat and Peyer’s patches (small intestine), wash out the luminal contents with cold PBS.
Wash the tissue pieces for 00:10:00 in 1 millimolar (mM) dithiothreitol (DTT)/PBS at Room temperature on a rocker to remove mucus, followed by a wash for 00:25:00 in 10 millimolar (mM) EDTA/30 millimolar (mM) HEPES/PBS at 37 °C on a platform shaker (180 rpm ) to remove epithelium.
35m
After a 00:02:00 wash in complete RPMI, digest the tissue in a 6-well plate for 01:30:00 in complete RPMI with 150 U/ml (small intestine) or 300 U/ml (large intestine) collagenase VIII (Sigma) and 150 µg/µL DNase (Sigma) in a cell culture incubator (5% CO2).
1h 32m
Pass the tissue digests through a 100 µm cell strainer and separate them by centrifugation (1200 x g for 00:20:00 ) using a 40/80% percoll gradient.
20m
Collect the immune cells at the 40/80% interface.
For the spleen, pass the tissue through a 100 µm cell strainer and incubate in red cell lysis buffer (Sigma) for 00:08:00 at Room temperature .
8m
Wash both spleen and intestine immune cells with 0.5% BSA/PBS before staining and fixation (eBioscience Foxp3 / Transcription Factor Staining Buffer Set).
Flow Cytometry
Use , CD16/32 antibody (eBioscience) for flow cytometry staining to block the non-specific binding to Fc receptors before surface staining.
Isolate the immune cells from intestinal lamina propria and stain with antibodies against the following markers:
| A | B | |
| Marker | Stain | |
| CD103 | PerCP-efluor710 | |
| CD11b | SuperBright645 | |
| CD11c | FITC | |
| CD19 | FITC | |
| CD3e | PE | |
| CD4 | APC | |
| CD45.2 | BV421 | |
| CD64 | APC-Cy7 | |
| CD8a | APC-e780 | |
| CSF1R | PE | |
| Ly6C | APC | |
| MHCII I-A/I-E | PE or PerCP-efluor710 | |
| TCRβ | PerCP-Cy5.5 |
For some panels, a lineage marker mix (Lin) contained TCRβ, B220, Ly6G and Siglec-F (PE-Cy7).
Discriminate the live and dead cells by Live/Dead Fixable Aqua Dead Cell Stain Kit (Invitrogen).