May 23, 2022

Public workspaceIntestinal Lamina Propria and Spleen Immune Cell Isolation

DOI
dx.doi.org/10.17504/protocols.io.dm6gpbxo5lzp/v1
  • 1California Institute of Technology
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DOI: dx.doi.org/10.17504/protocols.io.dm6gpbxo5lzp/v1
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
Abstract
This protocol details isolation of immune cells from intestinal lamina propria/spleen and flow cytometry.
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Isolation of Immune Cells from Intestinal Lamina Propria/Spleen
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 TemperatureOn 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
Wash the tissue pieces for Duration00:10:00 in Concentration1 millimolar (mM) dithiothreitol (DTT)/PBS at TemperatureRoom temperature on a rocker to remove mucus, followed by a wash for Duration00:25:00 in Concentration10 millimolar (mM) EDTA/Concentration30 millimolar (mM) HEPES/PBS at Temperature37 °C on a platform shaker (Centrifigation180 rpm ) to remove epithelium.

35m
Centrifigation
Wash
After a Duration00:02:00 wash in complete RPMI, digest the tissue in a 6-well plate for Duration01:30:00 in complete RPMI with Concentration150 U/ml (small intestine) or Concentration300 U/ml (large intestine) collagenase VIII (Sigma) and Concentration150 µg/µL DNase (Sigma) in a cell culture incubator (5% CO2).

1h 32m
Wash
Digestion
Pass the tissue digests through a Thikness100 µm cell strainer and separate them by centrifugation (Centrifigation1200 x g for Duration00:20:00 ) using a 40/80% percoll gradient.

20m
Centrifigation
Collect the immune cells at the 40/80% interface.
For the spleen, pass the tissue through a Thikness100 µm cell strainer and incubate in red cell lysis buffer (Sigma) for Duration00:08:00 at TemperatureRoom temperature .

8m
Incubation
Wash both spleen and intestine immune cells with 0.5% BSA/PBS before staining and fixation (eBioscience Foxp3 / Transcription Factor Staining Buffer Set).
Wash
Flow Cytometry
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:


AB
MarkerStain
CD103PerCP-efluor710
CD11bSuperBright645
CD11cFITC
CD19FITC
CD3ePE
CD4APC
CD45.2BV421
CD64APC-Cy7
CD8aAPC-e780
CSF1RPE
Ly6CAPC
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).