iPSC differentiation into Microglia

Day-2: Coat 3 wells of a 6 well pates with Matrigel. (1 hour coating also works)

Day-1 :

Bring iPSC maintenance to plate cell culture hood, remove the media and rinse once with PBS.

Now bring the Matrigel coated plate, aspirate the Matrigel and add 1.5mL of E8+Ri media. (3 well were coated on day0) Now plate 10 clumps in 1st well, 20 clumps in 2nd well and 30 clumps in 3rd well of the 6 well plate.* Shake the plate up and down and to sides, return the plate to the incubator.

Then add 1mL of 0.5micromolar (µM) EDTA to the well and leave in the incubator for about 0h 5m 0s. By this time, you will see colonies lifting from the plates. If not leave plate in the incubator for few more minutes.

Once the 50 to 60% of colonies come off the plate, bring plate into the hood and neutralize the reaction by adding 2mL of E8+Ri media.

Now gently swirl the plate to mix EDTA solution containing cells and E8+Ri media.

After swirling the plate pipet 1mL of the cells with 5 ml pipet into 15 ml falcon tube.

Spin down the cells at 1rcf.

After this spin, take aspirate the supernatant and gently tap the cell pellet.

Then add 3mL of E8+Ri media and tap gently once again to mix the clumps.

For counting the clump number take 5µL of this mix into a 96 well plate, add 100µL of E8 media. Gently tap the plate and count the colonies under the microscope. Repeat this in 3 wells of 96 well plate and average the clump number.

Day 0

Count the colonies in in each well, ideal colony number is between 10-30. It doesn’t have to be perfect number. Wells with as low as 5 colonies and max up to 30 can also be used. Anything above 40 should be avoided.

Choose on well with desired number of colonies and you can discontinue maintaining remaining wells.

After achieving desired colony number prepare Media A (2mL base media + 10µL supplement A).

Aspirate E8+Ri media and add 2mL Media A and leave plate in incubator for 48h 0m 0s.

Day 2

Add 1mL A (1mL base media + 5µL supplement A).

Day 3

Prepare Media B (2mL base media + 10µL supplement B).

Aspirate media A and add 2mL Media B.

Day 5, 7,9,10

supplement the cell with 1mL of Media B.

Day 12: Collection

By day 12 you will see lot of floating hematopoietic progenitor cells.

Collect hematopoietic progenitor cells by gently swirling the plate with a 5 ml pipet into 15 ml falcon tube.

Spin down the cells 3rcf.

Remove the supernatant and resuspend the cell pellet in Macrophage differentiation media. (RPMI+ 20% FBS+100ng/mL M-CSF).

Count the cell by using hemocytometer and plate 100,000 in one well of 6 well plate.

Day 12:

Plate 100,000 hematopoietic progenitor on Matrigel coated 6 well plate in 3 cytokine media.

On days 14,16,18,20 and 22 supplement with of 3 cytokine media. 1mL of 3 cytokine media.

Day 24

Collect 6mL of cells + media into 15 ml falcon by leaving 1mL conditioned media in the plate. Spin down at 3rcf and remove the supernatant.

Resuspend the pellet in 2mL of fresh 3 cytokine media and plate back into the same well containing conditioned media.

Day 26,28,30,32,34,36 supplement with of 3 cytokine media. 1mL of 3 cytokine media.

Day 37

Collect 6mL of cells + media into 15 ml falcon by leaving 1ml conditioned media in the plate.

Spin down at 3rcf and remove the supernatant.

Resuspend the pellet in 2mL of fresh 5 cytokine media and plate back into the same well containing conditioned media.

Day 39: supplement cells with 1mL of 5 cytokine media.

Day 41: Collect cells for experiment.