Apr 22, 2020

Public workspaceHubMap UF TMC Tissue Dissociation to Single Cell

DOI
dx.doi.org/10.17504/protocols.io.bd9vi966
  • 1University of Florida
  • Human BioMolecular Atlas Program (HuBMAP) Method Development Community
    Tech. support email: Jeff.spraggins@vanderbilt.edu
Marda Jorgensen
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DOI: dx.doi.org/10.17504/protocols.io.bd9vi966
Protocol Citation: Marda Jorgensen, Maigan Brusko 2020. HubMap UF TMC Tissue Dissociation to Single Cell. protocols.io https://dx.doi.org/10.17504/protocols.io.bd9vi966
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: March 26, 2020
Last Modified: October 02, 2020
Protocol Integer ID: 34837
Guidelines
Perform all steps using appropriate aseptic technique.
Materials
MATERIALS
ReagentForcepsGold BiotechnologyCatalog #F3001
ReagentScissorsCarl RothCatalog #HCT7.1
ReagentDMEMGibco - Thermo Fisher ScientificCatalog #11885
ReagentSterile deionized H2O
Reagent10X Collagenase/Hyaluronidase in DMEMSTEMCELL Technologies Inc.
ReagentMiltenyi C Tube
ReagentSterile Petri Dishes
ReagentDisposable Scalpels 10 Blade
ReagentFBS USDA OriginCatalog #25-550
ReagentPenicillin/StreptomycinCorningCatalog #30-002-CL
Reagent0.5M EDTABoston BioproductsCatalog #BM-150
ReagentAmmonium ChlorideFisher ScientificCatalog #A661-500
ReagentNaHCOFisher ScientificCatalog #S233-50
ReagentDisposable Filter Flask with PES Membrane
ReagentgentleMACS C-Tube Miltenyi BiotecCatalog #130-093-334
ReagentgentleMACS DissociatorMiltenyi BiotecCatalog #130-093-235
Reagent500um Cell StrainerpluriSelectCatalog #43-50500-01
Reagent100um Cell StrainerFisher ScientificCatalog #22363549
Reagent40um Cell Strainer Fisher ScientificCatalog #22363547
Reagent15ml Conical Tubes OlympusCatalog #21103
ReagentNexcelom Cellometer
ReagentNexecelom Cellometer Slides
ReagentNexecelom ViaStain AO/PI
Reagent1mL Cryovials Internally Threaded with O-ringOlympusCatalog #24-200P
ReagentCryostar CS10Merck MilliporeSigma (Sigma-Aldrich)Catalog #C2874
ReagentCool CellsbiocisionCatalog #BCS405G
ReagentLabXpert Label MakerBrady Worldwide, Inc.Catalog #060900
ReagentLabXpert Label Maker StickersBrady Worldwide, Inc.Catalog #X-120-492
Safety warnings
Use universal safety precautions when handling human samples and employ personal protective equipment (e.g., face mask with shield, gloves, lab coat or apron).
Preparation
Preparation
To make ACK Lysis Buffer:
Add 8.02 grams of NH4Cl, 0.84 grams of NaHCO, and 5uL of 0.5M EDTA into 1000mL of deionized water.
Filter buffer through 22um PES Filter. Aliquot in 50mL conicals and store at room temperature for long-term storage.
To make the cDMEM:
Add 50mL of FBS, 10mL of Penicillin/Streptomycin to 440mL of DMEM.
Filter through a filter flask and store at 4℃.
Procedure
Procedure
Obtain 1cm x 1cm x1cm tissue cube (thymus and spleen) or whole/partial lymph node from organ dissection protocol. Place in 15mL conical tube with 5mL cDMEM, retain on ice.
Prepare 10mL digest mix using 1mL of 10x Collagenase/Hyaluronidase in DMEM and 9mL dMEM with 5% FBS. Warm to 37C in water bath.
Mince tissue crosswise with two scalpels in sterile petri dish until tissue fragments are approximately 2 mm in all dimensions.
Transfer tissue fragments to C tube, incubate at 37℃ in shaking incubator for 30 minutes.
Place C tube on gentleMACS dissociator, run protocol mspln0201.
Transfer cell suspension to 50 ml conical over 500 uM mesh filter and wash over filter with DMEM until tube is full. Centrifuge 350 x g for 10 minutes beginning at RT, step down to 8℃.
Discard supernatant. Resuspend cell pellet in cold cDMEM, decant over 100 uM filter into new conical tube. Centrifuge 350 x g for 10 minutes, 8℃.
Discard supernatant and retain cell pellet. Resuspend in 10 ml cold HBSS, decant over 40 uM filter into new conical tube. Retain cell suspension on ice while performing cell count.
Take a 20ul aliquot for counting on a Nexcelom Cellometer.
Add 20ul of Viastain AO/PI to aliquot and mix thoroughly with pipette. Add 20ul to counting slide and count with “immune cells low RBC” with dilution factor 2.0. Record total yield and viability in case worksheet.
Centrifuge remaining cell suspension at 350 x g for 10 minutes at 8℃. Decant supernatant and dissociate cell pellet. Resuspend dropwise in Cryostor 10 at 25 million cells/ml.
Aliquot suspension to labeled cryovial and place in CoolCell. Transfer to -80℃ freezer for 18-24 hours prior to transfer to liquid nitrogen cryounit.