TITLE: Purification of Mouse Pancreatic Islets using Histopaque Gradient Centrifugation
AUTHORS: Nancy Smith, Haopeng Lin, Mourad Ferdaoussi, Patrick Macdonald

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol details the purification of mouse pancreatic islets using histopaque gradient centrifugation.</div></div>

[STEPS]
?. [Solution Prep - 1X Hanks Balanced Salt Solution (HBSS) - Sigma H6136]
?. [Solution Prep - 1X Hanks' Balanced Salt Solution (HBSS) + BSA - Sigma H6136 and Coating]
Measure out 900ml of room temperature H2O
[MiliQ water]
?. [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.
?. [Solution Prep - 1X Hanks' Balanced Salt Solution (HBSS) + BSA - Sigma H6136 and Coating]
Rinse original vial with water to remove traces and add to above.
?. [Solution Prep - 1X Hanks' Balanced Salt Solution (HBSS) + BSA - Sigma H6136 and Coating]
Add 0.35g sodium bicarbonate and stir until dissolved.
[Sodium Bicarbonate]
?. [Solution Prep - 1X Hanks' Balanced Salt Solution (HBSS) + BSA - Sigma H6136 and Coating]
Adjust pH to 7.4
?. [Solution Prep - 1X Hanks' Balanced Salt Solution (HBSS) + BSA - Sigma H6136 and Coating]
Bring solution to 1L
?. [Solution Prep - 1X Hanks' Balanced Salt Solution (HBSS) + BSA - Sigma H6136 and Coating]
Add 4ml of 30% BSA (equitech bio, inc 160928-0262) to the prepared 1X HBSS
?. [Solution Prep - 1X Hanks' Balanced Salt Solution (HBSS) + BSA - Sigma H6136 and Coating]
Store at 4°C
4 °C
?. [Solution Prep - Mouse Islet Culture Media]
Add FBS and Pen/Strep to bottle of RPMI  AB1500ml RPMI 1640 (11.1mM glucose)
Gibco 11875-119250ml FBS Canadian Origin&nbsp;
Gibco 12483-020
35ml Pen/strep (10000 Unit/ml/10000 ug/ml)Gibco 15140-122
AB1500ml RPMI 1640 (11.1mM glucose)
Gibco 11875-119250ml FBS Canadian Origin&nbsp;
Gibco 12483-020
35ml Pen/strep (10000 Unit/ml/10000 ug/ml)Gibco 15140-122
?. [Solution Prep - Mouse Islet Culture Media]
Store at 4°C
4 °C
?. [Gradient Protocol]
Once digest of pancreas in the waterbath is complete (see Mouse Pancreatic Islet Isolation protocol, Step 16)https://www.protocols.io/view/mouse-pancreatic-islet-isolation-sqaedseShake 50ml falcon tube 30 times.
?. [Gradient Protocol]
Centrifuge for 2 minutes at 339 g at 4°C
4 °C
?. [Gradient Protocol]
Remove supernatant
?. [Gradient Protocol]
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.
?. [Solution Prep - 1X Hanks' Balanced Salt Solution (HBSS) + BSA - Sigma H6136 and Coating]
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.
?. [Gradient Protocol]
Centrifuge for 2 minutes at 339 g at 4°C
4 °C
?. [Gradient Protocol]
Remove supernatant.  Add 10ml 1X HBSS+BSA, re-suspend pellet.  Bring up the volume to 50ml with 1X HBSS+BSA.
?. [Gradient Protocol]
Centrifuge for 2 minutes at 339 g at 4°C
4 °C
?. [Gradient Protocol]
Remove supernatant.  Add 10ml 1X HBSS+BSA, re-suspend pellet.  Bring up the volume to 50ml with 1X HBSS+BSA.
?. [Gradient Protocol]
Centrifuge for 2 minutes at 339 g at 4°C
4 °C
?. [Gradient Protocol]
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.
?. [Gradient Protocol]
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.
?. [Gradient Protocol]
Lay the tube at an angle on ice.  Add gradually 10ml of the Mouse Media to ensure that the two mediums do not mix.
?. [Gradient Protocol]
Centrifuge for 30 minutes at 339 g at 4°C with the brake turned off.
4 °C
Some centrifuges have an acceleration/deceleration speed setting.  Use slowest rate of speed for both acceleration and deceleration .
?. [Gradient Protocol]
Transfer the supernatant (both layers) into a new single 50ml falcon tube. Bring up to volume with 1X HBSS+BSA.
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.
?. [Gradient Protocol]
Centrifuge for 3 minutes at 339 g at 4°C.
4 °C
?. [Gradient Protocol]
Decant the supernatant.  Re-suspend the pellet in 10 ml of Mouse Media.
?. [Gradient Protocol]
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.
?. [Gradient Protocol]
Bring up volume to 50ml by adding 1X HBSS+BSA to falcon tube.