Feb 27, 2023
Splenocyte Preparation for Immune Reconstitution (Humanisation)
- Sandra Petrus-Reurer1,
- Kourosh Saeb-Parsy1
- 1University of Cambridge
Protocol Citation: Sandra Petrus-Reurer, Kourosh Saeb-Parsy 2023. Splenocyte Preparation for Immune Reconstitution (Humanisation) . protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7y6e9gwz/v1
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: February 27, 2023
Last Modified: February 27, 2023
Protocol Integer ID: 77679
Keywords: Mouse immune reconstitution, Humanisation, Splenocytes, splenocyte preparation for immune reconstitution, splenocyte preparation, immune reconstitution, mice
Funders Acknowledgements:
MRC UKRMP
Grant ID: G101985
Abstract
This protocol guides through the steps required to humanise mice with human splenocytes
Troubleshooting
Thaw vial (approx. 50x106 cells/vial) in water bath 37C for 2-3min (leave crystal)
Add cells into 10mL/vial of warm 50% RPMI media + 50% FBS
Centrifuge 7min at 300g
Combine all vials in 10mL RPMI media + 10% FBS
Centrifuge 7min at 300g
Resuspend all vials in 10mL RPMI media + 10% FBS + add DNAse-I (typically 40uL/mL if DNAse-I 1mg/mL) at 37C (incubator) for 15-20min (depending on the amount of clumps seen). Check and flick the tube every 5 min
Centrifuge 7min at 300g
Resuspend in 10mL RPMI media + 10% FBS and filter with 70um filter (white)
Count cells:
-If with Neubauer chamber: resuspend 1:2 (10uL cells + 10uL Trypan Blue); Calculation: average of cells per quadrant x 2 (dil) x 104 (cells/mL) x 10 (mL)
-If automatic counter: resuspend 1:10 (10uL cells + 90uL Trypan Blue); Calculation: average of two reads and take live cells x 5 (dil) x 10 (mL)
Resuspend pellet in right volume to: 50x106 cells/mL (10x106 cells/200uL=50,000 cells/uL) for NSG mice, or for NSG-dKO mice 100x106 cells/mL (20x106cells/200uL=100,000 cells/uL) with PBS + 2% FBS in universal tubes/epps
Freeze leftover cells (max 50x106cells/vial) with 10% DMSO in FBS (1mL per vial)
Take to the animal facility x2 the number of cells and volume needed per donor (in case there are losses due to clumping, dead volumes etc.)
Inject 200uL/mouse intraperitoneally
Follow humanization weekly by performing tail vein bleeds with heparin (applying red cell lysis buffer x3, 10min on ice) and flow cytometry for the immune markers: hCD45, mCD45, hCD19, hCD3, hCD8, hCD4, 7AAD