Feb 10, 2020

Public workspaceEndometrium dissociation with trypsin

DOI
dx.doi.org/10.17504/protocols.io.72dhqa6
  • 1Wellcome Sanger Institute
  • Vento-Tormo
    Tech. support email: rv4@sanger.ac.uk
Regina Hoo
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DOI: dx.doi.org/10.17504/protocols.io.72dhqa6
Protocol Citation: Regina Hoo, Roser Vento-Tormo 2020. Endometrium dissociation with trypsin . protocols.io https://dx.doi.org/10.17504/protocols.io.72dhqa6
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: October 08, 2019
Last Modified: February 10, 2020
Protocol Integer ID: 28453
Abstract
This protocol is for enrichment of epithelial glands on endometrium; following the step from "Endometrium dissociation with collagenase".


Guidelines
Human samples including tissue, blood and bodily fluids have the potential to harbour HG2 and Hazard Group 3 (HG3) organisms, specifically Blood Borne Viruses (BBVs,); and for brain tissue, CNS tissue and CSF, prions. In the UK we can work with such samples at CL2 on the condition that we do not intend to culture any of the organisms that might be contained in the samples and that the samples haven’t already been identified by tests or diagnosis as containing HG3 organisms.
Materials
MATERIALS
Reagent10% FBS RPMI
ReagentFBSInvitrogen - Thermo Fisher
ReagentRPMI 1640 MediumThermo Fisher ScientificCatalog #11875093
ReagentTrypsin-EDTA (0.25%) phenol redThermo Fisher ScientificCatalog #25200072
ReagentPBSInvitrogen - Thermo Fisher
ReagentDNAse IMerck MilliporeSigma (Sigma-Aldrich)Catalog #4716728001
Safety warnings
Samples are unscreened human tissues, please adhere to Biological Safety at Containment Level 2 work procedures.
Prepare Trypsin-EDTA-DNaseI mix:

ProductStockFinal volume (10 ml)Concentration
Trypsin-EDTA (0.25%) phenol red9.9 ml
DNaseI10mg/ml100 ul0.1 mg/ml

Following step 10 from "Endometrium dissociation with collagenase" protocol, where pieces of tissue are retained on the cell strainer.

Collect pieces retained on the filter by inverting the filter into a new 50 mL tub and adding 45 ml of PBS with a 1 mL pipette.
Centrifuge at 450 g for Duration00:05:00 together with collagenased sample. Discard supernatants.

Resuspend the pieces with Amount10 mL Trypsin/EDTA 0.25% and incubate for Duration00:20:00 at Temperature37 °C while shaking.

Add Amount20 mL of RPMI with 10% FBS.

Filter material slowly and carefully (very dens liquid) through a 100 μM strainer. Discard retained tissue.
Centrifuge at 500 rcf for Duration00:05:00 at Temperature4 °C . Discard supernatant.

Same step as 14 in "Endometrium dissociation with collagenase" protocol
{optional} Resuspend sample with Amount5 mL of RLB mix and incubate for Duration00:10:00

*RLB preparation: Dilute 10x RLB stock with water

After RBC lysis, add Amount10 mL of PBS and centrifuge at 500 rcf for Duration00:05:00

Resuspend cells in Amount1 mL of PBS in an Eppendorf.

Centrifuge at 500 rcf for Duration00:05:00 at Temperature4 °C .

Discard supernatant.
Resuspend with Amount250 µL of PBS (final volume depending on cell number)

Filter cells trough a cell strainer snap cap tube.
Count live cells using tryphan blue and haematocytometer.
{Optional} Resuspend cells in freezing medium to a concentration of 1x 107 cells and aliquot into cryogenic storage vials.