LRRK2 Cell lines

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I.D.	Description	Location	Grid #	Origin	Comments
HEK293 FLP IN TREX GFP-LRRK2-WT	LRRK2 WT inducibly expressing cells	Cryostore	33,34	Dario Alessi and Paul Davies	DMEM + 10% FBS+ LGlut + penstrep. Blastacidin at final concentration of 15 ug/ml and hygromycin at 200 ug/ml should be kept in the media at all times. Cells can be induced to express the construct with tetracycline or doxycycline at a final concentration of 1ug/ml. When cryo recovering the cells, it is best to defrost each into T75 flasks and conduct 1 passage without the blastacidin or hygromycin.
HEK293 FLP IN TREX GFP-LRRK2-G2019S	LRRK2 mutant inducibly expressing cells	35,36	Dario Alessi and Paul Davies
HEK293 FLP IN TREX GFP-LRRK2-KD	LRRK2 Kinase dead inducibly expressing cells	16,17	Dario Alessi and Paul Davies

I.D.

Description

Location

Grid #

Origin

Comments

HEK293 FLP IN TREX GFP-LRRK2-WT

LRRK2 WT inducibly expressing cells

Cryostore

33,34

Dario Alessi and Paul Davies

DMEM + 10% FBS+ LGlut + penstrep. Blastacidin at final concentration of 15 ug/ml and hygromycin at 200 ug/ml should be kept in the media at all times. Cells can be induced to express the construct with tetracycline or doxycycline at a final concentration of 1ug/ml.

When cryo recovering the cells, it is best to defrost each into T75 flasks and conduct 1 passage without the blastacidin or hygromycin.

HEK293 FLP IN TREX GFP-LRRK2-G2019S

LRRK2 mutant inducibly expressing cells

35,36

Dario Alessi and Paul Davies

HEK293 FLP IN TREX GFP-LRRK2-KD

LRRK2 Kinase dead inducibly expressing cells

16,17

Dario Alessi and Paul Davies

Production number	Description	Pseudotype	Functional titer (TU/ml)	Volume	Location	Comments
PD-12-197	LV-CMV-eGFPLRRK2 wild-type	VSV-G	4.50E+07	200 μl	Leila's Box -80°C	Leuven Viral Vector Core Website Determine the MOI for cell transduction with lentivirus constructs Multiplicity of Infection (MOI): Multiplicity of Infection is the number of transducing lentiviral particles per cell. It is highly recommended that for each new cell type to be transduced, a range of MOI be tested. This will determine the optimal amount of lentiviral supernatant needed for efficient transduction of each cell line used. To calculate: (Total number of cells per well) x (Desired MOI) = Total transducing units needed (TU). (Total TU needed) / (TU/ml reported on C of A) = Total ml of lentiviral particles to add to each well.
PD-12-289	LV-CMV-eGFP-LRRK2 G2019S	VSV-G	1.18E+07	100 μl
PD-12-279	LV-CMV-eGFP-LRRK2 G2019S/D1994A	VSV-G	1.22E+07	100 μl
PD-12-233	LV-CMV-eGFP (control)	VSV-G	2.13E+13	100 μl