Mar 27, 2025
Temporal Assessment of Immune Response
- Adrien Stanley1
- 1Allen Institute for Neural Dynamics
- Allen Institute for Neural Dynamics
Protocol Citation: Adrien Stanley 2025. Temporal Assessment of Immune Response. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8dqx6g2w/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: January 27, 2025
Last Modified: March 27, 2025
Protocol Integer ID: 119652
Keywords: Temporal assessment, Immune response, Vascular Access Button (VAB), Mouse catheter maintenance
Abstract
This protocol describes Vascular Access Button (VAB) catheter blood collection intended for later assessment of immune responses across time to different IP injected substances in awake mice. This protocol is preceded by “Mouse Catheter Maintenance” protocol.
Guidelines
- A maximum of 1% of the animal’s body weight in blood can be collected within a two-week period. Check mouse body weight before the start of the experiment to calculate the maximum volume permitted to take.
Materials
- OHAUS™ Compass CR Series ScaleFisher ScientificCatalog #01-922-341 .
- Container for weighing mouse.
- Dumont Forceps - Micro-Blunted TipsFine Science ToolsCatalog #11253-25
- BD Slip Tip Sterile SyringesFisher ScientificCatalog #309656
- BD® Needle 3/4 in. single use, sterile, 23 GBecton Dickinson (BD)Catalog #305143
- Insulin syringes, used for IP injection protocol (BD, Cat#: BD328438).
- Hamilton® syringe, luer tipMerck MilliporeSigma (Sigma-Aldrich)Catalog #24531
- Ethanol swabs (Uline, Cat#: 13-680-63).
- Hanna Pharmaceutical Supply Co., Inc. 0.9% SODIUM CHLOR. INJ.10MLFisher ScientificCatalog #NC9054335
- 500 units/mL heparinized 50% glycerol (Instech, Cat#: USP-HGS-500-10-VB-5).
- Hamilton® syringe, luer tipMerck MilliporeSigma (Sigma-Aldrich)Catalog #24531
- PinPort™ InjectorsInstech Laboratories, Inc.Catalog #PNP3M
- Sarstedt Inc SC Micro tube 1.3ml K3 EDTA, 100/pack, 10 packs/caseFisher ScientificCatalog #NC9414041
Safety warnings
- Wear required PPE consisting of lab coat and gloves.
- If performing terminal collection, ensure proper ventilation and airflow in a fume hood or air extraction system to reduce exposure.
- When handling syringes with needles, always ensure needles are pointed away from you to avoid any accidental sticks. Report any accidental sticks to EHS.
- Used needles and syringes should be disposed in a biohazard sharps container.
Ethics statement
This protocol has been approved by the Allen Institute Animal Care and Use Committee (IACUC).
PHS Assurance: D16-00781
AAALAC: Unit 1854
Before start
- Implant or acquire mice with VAB Instech implanted catheters from animal vendor.
- For at least 3 days prior to the experiment, handle mice so they become familiar with your scent and ensure minimal stress on the experiment day. Handling days do not need to be consecutive and can be done on or between maintenance days.
Protocol
NAME
Mouse VAB Catheter Maintenance
CREATED BY
Abbie Weaver
Prepare Flush and Lock Solution Syringes
Prepare Flush and Lock Solution Syringes
1m
1m
Prefill a flush syringe(s) (slip tip syringes Fisher Scientific catalog # 309656) with 50 µL of sterile saline (1 per mouse).
Wipe septum of vial with 70% ethanol swab and let air dry for 00:00:30 prior to puncturing septum.
30s
Use a 1 mL syringe with 23 Gauge needle attachment to withdraw ~100 µL saline solution to account for any dead volume when pinport injector is added.
- After withdrawal, point the syringe upward and away from you and gently flick the syringe to knock any bubbles up towards the needle.
Remove the needle attachment and attach pinport injector.
- Ensure the needle is pointed away from you and grip the base of the needle while pulling the syringe out. Then dispose of needle in sharps waste container.
- Prefill Hamilton syringe with 14 µL of 500 units/mL of heparinized 50% glycerol.
Wipe septum of vial with 70% ethanol swab and let air dry for 00:00:30 prior to puncturing septum.
30s
Use 1 mL syringe with 23-gauge needle attachment to pull ~100 µL of 500 units/mL of heparinized saline in 50% glycerol.
Remove plunger from Hamilton and insert needle of filled syringe into the back of the Hamilton syringe and fill until the liquid can be seen in a drop at the tip of the Hamilton syringe.
Remove needle from the back of the Hamilton syringe and reinsert plunger, ensuring no air is in the Hamilton syringe.
With the remainder of the 500 units/mL of heparinized saline in 50% glycerol in the 1 mL syringe, fill the pinport injector such that no air is present.
Connect the Hamilton syringe to the pinport injector, then lower the plunger to 14 µL .
- Use paper towel to dab away excess liquid from the pinport tip.
Between mice, fill a new pinport attachment with 500 units/mL of heparinized saline in 50% glycerol, then pull Hamilton syringe plunger up to 14 µL, then swap to the newly filled pinport attachment while ensuring no air in the syringe.
Prepare Mouse for Blood Collection
Prepare Mouse for Blood Collection
Place mouse cage in fume hood with prepared syringes and remove the lid.
Place mouse in container on scale and record body weight.
Do a health assessment per IACUC protocol and continue if mouse meets criteria to proceed.
Perform Catheter Blood Collection (Time Zero Collection)
Perform Catheter Blood Collection (Time Zero Collection)
Place mouse on grid of food hopper and remove cap from the Vascular Access Button (VAB) on the mouse's back.
Use empty withdrawal syringe to remove lock solution and a small amount of blood, discard in sharps waste container.
Use a new withdrawal syringe and take a maximum of 1% of the mouse's body weight divided by the number of non-terminal time points.
Note
For example, a 20g mouse receiving 4 blood collections means a maximum of 50 µL of blood collected per timepoint.
Remove withdrawal syringe and flush the catheter with 50 µL sterile saline using a flush syringe.
Remove flush syringe and attach Hamilton syringe, then fill the catheter with lock solution.
Remove Hamilton syringe and place cap on VAB.
Quickly expel drawn blood into a K3 EDTA coated tube and mix well then store On ice .
Inject Mouse with the Substance to be Tested
Inject Mouse with the Substance to be Tested
Refer to:
Protocol
NAME
Intraperitoneal Injection in an Adult MouseCREATED BY
Allen Institute
Return mouse to its cage until the next time point of blood collection.
Record the time of injection for reference for any future blood collection time points.
Repeat Catheter Blood Collection
Repeat Catheter Blood Collection
Repeat go to step #6 - go to step #12 for as many non-terminal blood collections timepoints as planned.
(Optional) Perform Terminal Blood Collection
(Optional) Perform Terminal Blood Collection
At the final time point (terminal collection), mouse will be anesthetized with isoflurane and blood can be collected via one of the following methods:
Catheter blood collection:
- While the mouse is immobile with shallow respiration and unresponsive to toe pinch, perform cervical dislocation.
- Repeat go to step #7 - go to step #12 , collecting as much blood as possible.
Cardiac puncture:
- Attach 23-gauge needle to empty 1 mL syringe
- Switch Isoflurane flow to nose cone and position the mouse supine
- While the mouse is immobile with shallow respiration and unresponsive to toe pinch lay mouse on its back and gently insert needle below mouse's sternum and through the diaphragm targeting the heart.
- Gently pull the plunger and if blood is seen, continue pulling until syringe is filled. If blood is not seen, adjust the position of the needle and try again.
After completion of experiment, blood can be processed with the protocol “Processing blood intended for Olink assay.”