Oct 06, 2022

Public workspaceSplenocyte 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://protocols.io/view/splenocyte-isolation-from-mice-chigt4bw
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
Abstract
Simple protocol for isolation of spleenocytes from mouse spleen.
Protocol materials
ReagentFCS (Fetal Calf Serum) Life Technologies
Step 4
ReagentEDTA disodium dihydrateAbblisCatalog #AB1011793
Step 4
Reagent0.4% Trypan blue solution
Step 13
ReagentEasySep™ Mouse NK Cell Isolation KitSTEMCELL Technologies Inc.Catalog ## 19855
Step 16
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
Amount50 mL FACS buffer
Reagent1X PBS (Phosphate-buffered saline )
ReagentFCS (Fetal Calf Serum) Life Technologies 2 %
ReagentEDTA disodium dihydrateAbblisCatalog #AB1011793 1 mM

Spin down for 5 min Duration00:00:00
Centrifigation400 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
Amount1 mL per spleen
Duration00:05:00
TemperatureRoom temperature

5m
Incubation
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
Amount50 mL FACS buffer

Centrifigation400 x g, 4°C, 00:05:00

5m
Pour off supernatnat
Resuspend in 5 mL of FACS buffer per spleen
Amount5 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
Reagent0.4% Trypan blue solutionContributed by users


Centrifigation400 x g, 4°C, 00:05:00

5m
Resuspend cells to obtain a concentration of 1 x 10^8 cells/mL
Concentration1x10^8 Mass Percent

Continue with ReagentEasySep™ Mouse NK Cell Isolation KitStemcell TechnologiesCatalog ## 19855 for isolatoin of primary mouse NK cells.