Tissue culture protocol for INS-1 cells and derived lines


Growth medium:

 

RPMI-1640 medium with:

                                                            10% fetal calf serum

            And INS-supplement              10mM Hepes

                                                            2mM L-glutamine

                                                            1mM Sodium-pyruvate

                                                            0.05 mM 2-mercaptoethanol

 

Formulation for INS-supplement

 

100x Stock solution                                                    Final Concentration

HEPES                                    119g                            10mM
Glutamine                               14.6g                           2mM

Na-Pyruvate                            5.5g                             1mM

2-mercaptoethanol                  0.176mL                      0.05mM

 

Dissolve above ingredients in 400 mL RPMI-1640, ph to 7.2 and bring to final volume of 500 mL with RPMI-1640. Filter sterilize and aliquot. Store aliquots at -20C.

Add 5.5 mL of 100x stock to 500mL RPMI. Add 50 ml of fetal calf serum.

 

Alternative preparation of 50x INS-supplement:.

 

Glutamate solution (200mM)                                                  100mL

Na-pyruvate solution (100mM)                                              100mL

2-mercaptoethanol                                                                  35.2 uL

 

Mix above solutions, filter sterilize (0.2 um filter) store aliquots at -20 C.

 

To make growth medium add 11 mL of 50x INS-supplement, add 5mL HEPES-solution (1M) and 50 mL fetal calf serum to 500 mL of RPMI-1640

 

Thawing Cell Lines:

 

Vial should be sufficiently sprayed with 70% ethanol and be thawed in a 37C water-bath until a small piece of frozen medium remains (2-3 mins).

Contents of vial should be transferred to a T-75 tissue culture flask filled with a 30-40mL of growth medium. Normally cells attach within 24 hours and we see >85% viability after thawing.

 

Sub Culturing Procedures

 

Volumes used in this protocol are for 75 cm2 flasks.

  1. Remove and discard culture medium
  2. briefly rinse the cell layer with 15mL PBS w/o Ca2+ and Mg2+ .
  3. Add 2-3 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (2-4 mins). Do not agitate the cells by hitting or shaking the flask.
  4. Add 10 mL of culture medium and mix gently.
  5. Add aliquot of cell suspension to new flask. (Recommended sub-culture ration 1:4 to 1:8). 30-40 mL culture medium /75cm2‑ flask, medium should be changed after 3 days and cells sub-cultured when confluent     

 

Glucose Stimulated Insulin Secretion

 

For best results in glucose stimulated insulin secretion, cells need to be confluent.


We seed cells at a density of 0.5x106 / well in 1mL medium for 24-well plate, or 1x106/ well in 2 mL of medium for 12-well plates.

After 2 days we change the medium and perform assay on day 3. (832/13 and 833/15) Lines 832/1 and 832/2 grow slightly slower and we change medium day 3 and perform the assay on day.

 

*** Alternate Method***

Plate the 832/13 cells at a 1:2 or a 1:4 split by area. The cells split 1:2 will be ready after 3 days and the 1:4 will be confluent after 4 days. Media is changed 24 hr before the GSIS.

The glucose stimulated insulin secretion is performed in HBSS (Hepes Balanced Salt Solution). For formula see: hohmeier et al. 2000, Diabetes 49:424-430

 

For the assay we wash the cells twice with HBSS. The first wash is just a quick rinse, for the second wash we leave the HBSS on for 2 hr.

Caution: The cells do not attach very firm to the plates and will wash off very easy, if solution are added with too much force.

 

After 2 hours, the secretagogues diluted in HBSS are added for 2hr. For 24-well plate we add a1ml/well and 1.5-2ml?well to the 12-well plate.

After 2 hours we remove the solution for a human insulin RIA. Keep in mind that these cells secrete a mixture of rat and human insulin and an insulin RIA which is cross reactive between these two species will give you higher sensitivity – especially if you want to adapt the assay for a 96-well format.

 

We commonly use HBSS + 3mM glucose as a basal insulin secretion level and HBSS + 15mM glucose as stimulated level.

HBSS+3mM glucose + 200 uM diazoxide will serve as a control for true basal level and should be the same as HBSS + 3mM glucose.

HBSS +15mM glucose + 100uM IBMX will give you a maximum stimulation.