Oct 06, 2022

Splenocyte isolation from mice

This protocol is a draft, published without a DOI.
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Protocol Citation: sffschn 2022. Splenocyte isolation from mice. protocols.io https://dx.doi.org/
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 06, 2022
Last Modified: October 06, 2022
Protocol  Integer ID: 70952
Keywords: Mouse, Organ processing, Spleen, splenocyte isolation from mice simple protocol, isolation of spleenocyte, splenocyte isolation, spleenocyte, mouse spleen, mice simple protocol, mice
Abstract
Simple protocol for isolation of spleenocytes from mouse spleen.
Protocol materials
1X PBS (Phosphate-buffered saline )
FCS (Fetal Calf Serum) Life Technologies
EDTA disodium dihydrateAbblisCatalog #AB1011793
0.4% Trypan blue solution
EasySep™ Mouse NK Cell Isolation KitSTEMCELL Technologies Inc.Catalog ## 19855
Collect spleens in some PBS

Place spleen on a 70 µm mesh filter ontop of a 50 mL Falcon

Equipment
70 µm Cell Strainer
NAME
Cell Strainer
TYPE
Falcon
BRAND
352350
SKU
https://ecatalog.corning.com/life-sciences/b2c/US/en/Cell-Culture/Cell-Culture-Accessories/Cell-Strainers/Falcon®-Cell-Strainers/p/352350?clear=true
LINK
White, Sterile, Individually Packaged
SPECIFICATIONS

max. 2 spleens per filter
pass through 70 µm mesh filter using a pistol of a 2 mL syringe
wash with FACS buffer and top up to 50 mL
50 mL FACS buffer
1X PBS (Phosphate-buffered saline )
FCS (Fetal Calf Serum) Life Technologies 2 %
EDTA disodium dihydrateAbblisCatalog #AB1011793 1 mM

Spin down for 5 min 00:00:00
400 x g, 4°C, 00:05:00

Equipment
Eppendorf™ 5810R Centrifuge
NAME
Centrifuge
TYPE
Eppendorf
BRAND
02-262-8187
SKU
https://www.fishersci.com/shop/products/eppendorf-5810r-centrifuge-rotor-packages-16/022628187
LINK

5m
Pour off supernatant
Add 1 mL of RBC lysis buffer per spleen and incubate for 5 min at RT
1 mL per spleen
00:05:00
Room temperature

5m
Add a bit of FACS buffer and pass through new 70 µm mesh filter into a new 50 mL Falcon
Fill up to 50 mL
50 mL FACS buffer

400 x g, 4°C, 00:05:00

5m
Pour off supernatnat
Resuspend in 5 mL of FACS buffer per spleen
5 mL FACS buffer per spleen

Count cells
Equipment
Neubauer Improved Haemocytometer Counting Chamber
NAME
Counting Chamber
TYPE
Hawksley
BRAND
AC1000
SKU
https://hawksley.co.uk/products/neubauer-improved-haemocytometer-counting-chamber
LINK
Void depth: 0.1 mm Counting Area: 1mm²
SPECIFICATIONS
0.4% Trypan blue solution


400 x g, 4°C, 00:05:00

5m
Resuspend cells to obtain a concentration of 1 x 10^8 cells/mL
1x10^8 cells/mL

Continue with EasySep™ Mouse NK Cell Isolation KitSTEMCELL Technologies Inc.Catalog ## 19855 for isolatoin of primary mouse NK cells.