Human Islet Isolation Media Preparation

A	B	C	D	E
Hank's Balanced Salt Solution - HBSS (perfusion, rinse, priming solutions) (10L)
Reagent	Concentration	Weight/Volume	Supplier	Catalogue #
HBSS Powder	9.51 g/L	1 bottle	Mediatech/Corning	55-022-PB
CaCl2 (anhydrous)	3.60 mM	4.0g	MP Biochemicals LLC	15350290
MgSO4 (anhydrous)	0.81 mM	0.98g	VWR Life Science	97061-438
NaHCO3	4.2 mM	3.5g	Fisher Scientific	S233-500
HEPES	10mM	23.83g	Fisher Scientific	BP310-1
Penicillin/Streptomycin	100 U/ml penicillin 100 μg/ml streptomycin	50ml	Lonza	09-757F

A	B	C	D	E
Medium 199 - M199 (Wash media) (10L)
Reagent	Concentration	Weight/volume	Supplier	Catalogue #
M199 powder	9.41g/L	1 bottle	Mediatech/Corning	90050PB
NaHCO3	26 mM	22.0g	Fisher Scientific	S233-500
HEPES	10 mM	23.83g	Fisher Scientific	BP310-1
Penicillin/Streptomycin	100 U/ml penicillin 100 μg/ml streptomycin	50ml	Lonza	09-757F

A	B	C	D	E
Eagle Modified Minimal Essential Medium - EMEM (Dilution Media) (10L)
Reagent	Concentration	Weight/volume	Supplier	Catalogue #
EMEM powder	9.23g/L	1 bottle	Mediatech/Corning	90009BP
NaHCO3	26 mM	22.0g	Fisher Scientific	S233-500
HEPES	10 mM	23.83g	Fisher Scientific	BP310-1
Penicillin/Streptomycin	100 U/ml penicillin 100 μg/ml streptomycin	50ml	Lonza	09-757F

Prepare the HBSS, M199, and EMEM solutions outlined in step 1 of the Stock Media Preparation table using the following directions:1. Dispense 9L of Milli-Q (18mΩ) water in to the carboy

1. Store overnight at 4°C to allow to come to temperature.

2. Using the stirrer add the media powder to the water and allow to go into solution.

3. Add the powdered supplements and Penicillin/Streptomycin to the appropriate media based on the above tables and allow to stir into solution.

4. Stir the solution for 0h 30m 0s

5. Store the prepared solution overnight at 4°C to allow all powders to go into solution

6. Stir the solution for 0h 30m 0s

7. Calibrate the pH meter using the pH control buffers

8. Adjust the pH level to 7.4 using the NaOH and/or HCl solutions.

9. Bring to volume with the appropriate amount of Milli-Q water.

Sterile filter using the peristaltic pump, the tubing (silicon 25 gauge) and the capsule filter (Nylon Capsule filter 0.22µm membrane)  into the appropriate sterilized media bottle. 1. Store the filtered media at 4°C until needed for supplementation prior to isolation.

A	B	C	D	E
Nicotinamide Solution - 1M (1L)
Reagent	Concentration	Weight/volume	Supplier	Catalogue #
Nicotinamide powder	1 M	122.12	Sigma Aldrich Canada Co	N0636
HEPES	10 mM	2.383g	Fisher Scientific	BP310-1

Dispense 0.9L of Milli-Q water in to the carboy. 1. Store overnight at 4°C to allow to come to room temperature.

1. Using the stirrer add the reagent powder into the water based on the table (Step 4) and allow to stir into solution.
2. Stir the solution for 0h 30m 0s
3. Calibrate the pH meter using the pH control buffers
4. Adjust the pH level to 7.4 using the NaOH and/or HCl solutions
5. Bring to volume (1L) with the appropriate amount of Milli-Q water.
6. Filter sterilize using a bottle top filter (0.22μM) into a sterile 1L bottle.

Preparation of dithizone 1. Weigh out 0.2g of dithizone powder into a 50ml conical tube.

1. Add 6mL of DMSO and mix until the powder is in solution.
2. Bring the resulting dithizone solution to 40ml total volume with HBSS and mix.
3. Transfer the dithizone solution to a 60cc syringe with a 0.45µm nylon filter.

Use

1. For every ml of islet suspension add an equal amount of the prepared dithizone solution must be added to the sample.
2. For visualization of staining add another 2mL of HBSS to dilute the stain and reduce the background colour.
3. Alternately, 100µL of islet suspension, 100µL dithizone and 200µL HBSS.

To prepare the working solution supplement each of the listed stock medias with the indicated supplements.

A	B	C	D	E	F	G	H
	Total volume per bottle	BSA 30%	HBSS	EMEM	M199	Nicotinamide (1M)	Betadine
	(ml)	(ml)	(ml)	(ml)	(ml)	(ml)	(ml)
SOLUTION
M199 (aliquot into 12 x 100ml)	1172	160 (4.0%)	-----	-----	1000	12	-----
Dilution - make 3 bottles	2020	-----	-----	2000	-----	20	-----
Priming solution	1000	-----	1000	-----	-----	-----	-----
Wash 1 - split into 2x 550ml	1105	95 (2.5%)	-----	-----	1000	10	-----
Wash 2 - make 5 bottles	1055	45 (1.125%)	-----	-----	1000	10	-----
Cannulation	500	-----	500	-----	-----	-----	-----
Perfusion Solution	350	-----	350	-----	-----	-----	-----
Decon 1	300	-----	250	-----	-----	-----	50
Decon 2	250	-----	250	-----	-----	-----	-----
Decon 3	250	-----	250	-----	-----	-----	-----

Collection tubes for collection of tissue during the purification step.

A	B	C	D	E	F	G
Gradient Collection tubes
tube #	1	2	3	4	5	6
ml of wash 2 solution	100	150	200	200	225	225
tube #	7	8	9	10	11	12
ml of wash 2 solution	225	225	225	225	225	150

     Media Total per bottle CMRL BSA 30%  ITS (100x) Glutamax Pen/strep   Culture Media 521 500 8.5 (0.5%) 5 5 2.5   

A	B	C	D	E
Density Gradients
	lympholyte 1.100g/ml	UW sol'n. 1.042 g/ml	Volume (ml)	Final Density g/ml
High density	77	64	141	1.074
Low density	52	88	140	1.064

Preparation of the human islet culture media

Prepare the culture media by supplementing CMRL by the following table. Following these additions to the culture media, filter sterilize the resulting solution through a 0.22 µm Stericup Sterile Vacuum Filtration System.

A	B	C	D	E	F
	CMRL 1066	Bovine serum albumin 30%	Insulin-Transferrin-Selenium (100x)	Gibco® GlutaMAX Supplement	Penicillin-Streptomycin Mixture
	(ml)	(ml)	(ml)	(ml)	(ml)
Culture media	500	8.5	5	10	2.5

CMRL 1066 - Corning Catalogue # 15110CVBovine Serum Albumin 30% v/v - Equitech Bio Inc. Catalogue # BAL62Insulin-Transferrin-Selenium (100x) - Corning Catalogue # 25800CRGibco® GlutaMAX™ Supplement (100x) - ThermoFisher catalogue # 35050061Penicillin-Streptomycin Mixture - Lonza Catalogue # 09-757F