Jun 19, 2020
Version 2
Zebrafish Embryo Dissociation for MACS V.2
- Sam Wattrus1,2
- 1Harvard University;
- 2Boston Children's Hospital
Protocol Citation: Sam Wattrus 2020. Zebrafish Embryo Dissociation for MACS. protocols.io https://dx.doi.org/10.17504/protocols.io.bhquj5ww
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: June 19, 2020
Last Modified: June 19, 2020
Protocol Integer ID: 38388
Abstract
Method of zebrafish embryo dissociation and microbead binding for magnetic enrichment by MACS.
Guidelines
All steps here may should be optimized for a given transgenic line, and particular stage of development (where cells are more or less sticky, more or less rare, etc.) Steps here were optimized for harvesting cells from 1- 3 dpf embryos expressing either kdrl:hCD4 or runx1+23:hLNGFR.
If assessing efficacy of magnetic enrichment, cross lines into fluorescent reporters for analysis by flow cytometry.
Materials
MATERIALS
Trypan blueBio Basic Inc.Catalog #TT1140.SIZE.10g
Hemocytometer (Neubauer)
Razor blade
Ice
DMEMInvitrogen - Thermo Fisher
Falcon® 5 mL Round Bottom Polystyrene Test Tube, with Cell Strainer Snap CapCorningCatalog #352235
Thermomixer
PBS
FBS
Tumor Dissociation Kit humanMiltenyi BiotecCatalog #130-095-929
MACS Buffer (0.5% BSA 2mM EDTA in PBS)
MicrobeadsMiltenyi Biotec
Other enzyme mixes for dissociation may work as well, however they must preserve the epitopes used for microbead binding. Miltenyi provides a list of preserved epitopes for each of their enzyme kits online. This kit will preserve human CD4 and human LNGFR. Enzymes should be reconstituted and aliquoted at -20C.
Before start
Acquire or derive a transgenic line expressing a human surface peptide for antibody binding, and purchase the appropriate human microbead kit.
Prepare enzyme mix by adding 200ul Enzyme H, 100ul Enzyme R, and 25ul Enzyme A to 4.7mL DMEM on ice.
Tricaine and transfer embryos in minimal E3 to plate lid. Remove excess E3 (can be done effectively by blocking embryos with razor blade and wicking up water behind the blade with a Kimwipe). Chop embryos in plate lid. Make sure blade is in full contact with bottom surface, rotating the plate lid periodically.
Recover in 1.5mL enzyme mix to Eppendorf tube.
Vortex and incubate for 15-20 minutes at 37C in thermomixer. Vortex and pipet up and down vigorously every ~5 minutes until sample is dissociated.
Add FBS to 10% to quench (~150ul).
Filter through 40um blue cap FACS tubes on ice.
Transfer to fresh Eppendorf tubes and spin down 4 minutes @ 800rpm (tabletop centrifuge – aprox 60xg).
Carefully remove and discard supernatant. Resuspend cells in 1mL cold PBS. Take 1ul and add to 99ul Trypan blue in an Eppendorf to count a 1:100 dilution.
Spin down in tabletop centrifuge for 5 minutes at 300xg.
While cells are spinning down, mix 1:100 dilution and use 10ul to count total cell number on a hemocytometer.
Remove and discard supernatant, resuspend in 60ul MACS Buffer per 107 total cells. Add 20ul Microbeads per 107 total cells.
Mix well and incubate for 15 minutes @ 4C protected from light.
Wash by adding 1-2 mL MACS Buffer and centrifuge 5 minutes at 300xg, aspirate supernatant completely.
Resuspend up to 108 cells in 500ul of MACS Buffer.
Proceed to magnetic separation.
These cells are now compatible for use with either the AutoMACS Pro separator or manual column separation.
If proceeding to single cell RNA sequencing after magnetic enrichment, cells should be washed with PBS/0.5% BSA, spun down and resuspended before counting on a hemocytometer. EDTA in the MACS buffer can inhibit reverse transcription, so this should be removed.