Apr 14, 2020
Purification of Mouse Pancreatic Islets using Histopaque Gradient Centrifugation
- Nancy Smith1,
- Haopeng Lin1,
- Mourad Ferdaoussi1,
- Patrick E Macdonald1
- 1University of Alberta
- CIRTNR2FIC
External link: http://www.bcell.org
Protocol Citation: Nancy Smith, Haopeng Lin, Mourad Ferdaoussi, Patrick E Macdonald 2020. Purification of Mouse Pancreatic Islets using Histopaque Gradient Centrifugation. protocols.io https://dx.doi.org/10.17504/protocols.io.u7ueznw
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 31, 2018
Last Modified: April 14, 2020
Protocol Integer ID: 17364
Keywords: Histopaque, islets, isolation, gradient, mouse, pancreas,
Abstract
This protocol details the purification of mouse pancreatic islets using histopaque gradient centrifugation.
Materials
MATERIALS
Hank Balanced Salt Solution 1XMerck MilliporeSigma (Sigma-Aldrich)Catalog #H6136
Albumin Bovine Serum (30%)equitech bio, inc.Catalog #160928-0262
Histopaque 1077Merck MilliporeSigma (Sigma-Aldrich)Catalog #1077-1
RPMI 1640 (11.1mM glucose)Gibco - Thermo Fisher ScientificCatalog #11875-119
FBS Canadian OriginGibco - Thermo Fisher ScientificCatalog #12483-020
Cell dissociation sieveMerck MilliporeSigma (Sigma-Aldrich)Catalog #CD1-1KT
50ml Falcon tubesCorningCatalog #352070
30ml syringe Becton Dickinson (BD)Catalog #302832
18G needleBecton Dickinson (BD)Catalog #305196
Penicillin StreptomycinInvitrogen - Thermo FisherCatalog #15140 122
Sodium bicarbonateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S5761
Before start
1X Hanks' Balanced Salt Solution (HBSS) + BSA, Coating and Mouse Islet Culture Media should be prepared in advance and should be cold before starting gradient steps.
Solution Prep - 1X Hanks' Balanced Salt Solution (HBSS) + BSA - Sigma H6136 and Coating
Solution Prep - 1X Hanks' Balanced Salt Solution (HBSS) + BSA - Sigma H6136 and Coating
Measure out 900ml of room temperature H2O
900 mL MiliQ water
Solution Prep - 1X Hanks' Balanced Salt Solution (HBSS) + BSA - Sigma H6136 and Coating
Solution Prep - 1X Hanks' Balanced Salt Solution (HBSS) + BSA - Sigma H6136 and Coating
While gently stirring the water, add the powdered medium. Stir until dissolved. DO NOT HEAT.
Rinse original vial with water to remove traces and add to above.
Add 0.35g sodium bicarbonate and stir until dissolved.
0.35 g Sodium Bicarbonate
Adjust pH to 7.4
Bring solution to 1L
Take 34ml of the prepared 1X HBSS and add 6ml of 30% BSA (equitech bio, inc 160928-0262) in a 50ml falcon tube. This is to be used for coating.
Add 4ml of 30% BSA (equitech bio, inc 160928-0262) to the prepared 1X HBSS
Store at 4°C
4 °C
Solution Prep - Mouse Islet Culture Media
Solution Prep - Mouse Islet Culture Media
Add FBS and Pen/Strep to bottle of RPMI
| 500ml RPMI 1640 (11.1mM glucose) | Gibco 11875-119 | |
| 50ml FBS Canadian Origin | Gibco 12483-020 | |
| 5ml Pen/strep (10000 Unit/ml/10000 ug/ml) | Gibco 15140-122 |
Store at 4°C
4 °C
Gradient Protocol
Gradient Protocol
Once digest of pancreas in the waterbath is complete (see Mouse Pancreatic Islet Isolation protocol, Step 16)
Shake 50ml falcon tube 30 times.
Bring up volume to 50ml by adding 1X HBSS+BSA to falcon tube.
Centrifuge for 2 minutes at 339 g at 4°C
02:00:00
4 °C
Remove supernatant
Re-suspend pellet in 20ml of 1X HBSS+BSA. Pour re-suspended pellet through #60 screen (cell dissociation sieve, Sigma CD1-1KT) that has been rinsed with coating. Using a 30ml syringe filled with 1X HBSS+BSA and 18G needle rinse screen (use about 20ml). Return islets to 50ml tube and rinse the beaker with the remaining 10ml of 1X HBSS+BSA, add it to the tube.
Centrifuge for 2 minutes at 339 g at 4°C
02:00:00
4 °C
Remove supernatant. Add 10ml 1X HBSS+BSA, re-suspend pellet. Bring up the volume to 50ml with 1X HBSS+BSA.
Centrifuge for 2 minutes at 339 g at 4°C
02:00:00
4 °C
Remove supernatant. Add 10ml 1X HBSS+BSA, re-suspend pellet. Bring up the volume to 50ml with 1X HBSS+BSA.
Centrifuge for 2 minutes at 339 g at 4°C
02:00:00
4 °C
Remove supernatant. Leave tube upside down on papertowel to drip dry (30 seconds). Wipe the interior of the tube with a Kimwipe using a long forceps (do not touch the pellet). Leave tubes upright on ice.
Re-suspend the pellet with 5ml of Histopaque 1077 (Sigma 1077-1 500ml) with pipette that has been rinsed with coating. Add another 5ml of Histopaque and mix using coated pipette.
Lay the tube at an angle on ice. Add gradually 10ml of the Mouse Media to ensure that the two mediums do not mix.
Centrifuge for 30 minutes at 339 g at 4°C with the brake turned off.
30:00:00
4 °C
Note
Some centrifuges have an acceleration/deceleration speed setting. Use slowest rate of speed for both acceleration and deceleration .
Transfer the supernatant (both layers) into a new single 50ml falcon tube. Bring up to volume with 1X HBSS+BSA.
Note
Keep tubes with the pellet on ice. If islets are not in the the supernatant, they may be in the pellet and can be recovered by hand picking in Mouse Islet Media.
Centrifuge for 3 minutes at 339 g at 4°C.
03:00:00
4 °C
Decant the supernatant. Re-suspend the pellet in 10 ml of Mouse Media.
Hand pick islets if desired or necessary. Incubate islets in Mouse Islet Media at 37°C with 5% CO2 until ready for use in experiment.