PLB-985 propagation and differentiation

Medium: RPMI 1640 medium, gibco

Supplements: antibiotics (P/S, 25030–024, gibco), L-glutamine (Q, 15140–122, gibco), dimethylformamide (DMF, D4551, Sigma)

Propagation of cells

1.Thaw aliquot in waterbath, transfer to 15ml falcon, add 5ml of RPMI containing 20% (v/v) FCS, P/S and Q

2.Centrifuge at 300xg for 10min at room temperature, aspirate supernatant

3.Resuspend in 5ml RPMI 20% FCS, P/S and Q

4.Repeat washing step, resuspend in 5ml RPMI 20% FCS, P/S and Q

5.Transfer to a T25 flask and incubate at 37°C 5% CO2 overnight

6.Change medium (spin cells at 300xg for 5min, resuspend in 5ml RPMI 20 % FCS, P/S and Q)

7.Incubate in T25 flask for 2-3 days

8.Change medium to RPMI 10% FCS, P/S and Q, use this as the standard culture medium

(Freeze new aliquots as soon as possible! Use cells at 1x10^7/ml in FCS 10% (v/v) DMSO for freezing)

9.Split cells every 3-4 days to a desired density (roughly 0.5 – 0.8 x10^6/ml)

10.Thaw a new aliquot every 6-8 weeks

Differentiation of cells

1.At d0, centrifuge cells at 300xg for 5min

2.Resuspend in 1-2ml of differentiation medium (RPMI 2.5% (v/v) FCS, 0.5% (v/v) DMF, P/S and Q), count cells

3.Adjust concentration to 0.4 x 10^6 cells/ml

4.Transfer to desired volume (generally, 1 well of a 6 well plate containing 3ml (=1.2x10^6 cells) will result in around 5x10^6 differentiated cells at day 7) -> this is day 0 of differentiation

5.On day 4, add fresh differentiation medium to the cells (add 2ml to a 6 well or adjust accordingly for other volumes)

6.On day 7, collect cells into 15ml falcon tubes, centrifuge at 300xg for 5min, resuspend in 1ml of differentiation medium

7.Layer carefully onto 2ml Histopaque-1077 (Sigma) in 15ml falcon tubes using pasteur pipettes

(This Histopaque step is to remove dead cells and debris)

8.centrifuge for 20 min at 800xg, brakes off (important to use reduced acceleration and braking of the centrifuges, the amount of reduction depends on the model)

9.Collect cells on top of the Histopaque layer with a pipet, transfer to new falcon tube

10.wash with 2ml assay medium (RPMI without phenol red (gibco), 1%FCS (v/v), 0.5%DMF (v/v) and Q. Note that no antibiotics are used in assay medium to allow experiments involving bacterial phagocytosis and killing)

11.resuspend in 1ml assay medium, count

12.Adjust to desired density for experiments (usually 1 x 10^6/ml for experiments in 96 well plates – 100 microliters per well = 10^5 cells)