transduction protocol for AML CD34+ cells

Lentiviral  AML transduction protocol:

 

Day 1

-         Isolate AML CD34 cells with MACS column or sort on Facs and incubate (0.5 million /mL) in Gartners with IL-3, G-CSF and TPO (each 20 ng/mL) until ready, 37ºC, 5% CO2

-         Make  tubes 1 and 2 (see addition to….)

 

For every transduction you need  3 (or more)groups:

Group 1: no virus control (for procedure and Facs gates)

Group 2: control vector

Group 3: vector with gene of interest (GOI)

In 96 wells forma t: add 50.000 CD34 AML cells per well for 5 or 10 wells (total 250.000 to 500.000 cells) (for T25 co-culture, use total of no less then 200.000 cells) in viral transduction supernatant 

 

 

Example:

Group 1                                        Group 2                                  Group 3

 

500 µl HPGM                               500µl virus sup control 500 µl virus sup GOI

500.000 cells                                500.000 cells                          500.000 cells

50 µl FCS-GF                               50 µl FCS-GF                                     50 µl FCS-GF

5 µl FCS-poly                                5 µl FCS-poly                         5 µl FCS-poly

10x 50 µl   plate                             10x 50 µl plate                         10x 50 µl plate

 

ON 37ºC, 5% CO2 (round 1)

 

Day 2

-         repeat procedure in morning (round 2). Make virus sups  (or HPGM) with FCS and GF and polybrene (again in 500 µl), see above

-         add 50 µl to each well, (no wash, simply add)

-         evening:  repeat procedure (round 3)

-         coat T25 with 0.1 % gelatin

-         plate MS5, till confluency next morning

 

Day 3

-         Facs with beads for efficiency and count

-         Plate equal amounts of cells on MS5 in Gartners with IL-3, G-CSF and TPO (each 20 ng/mL).

-         I usually also plate the non virus control. This because the procedure might affect the AML and in this way you control for that. On the other hand, you  can use these to set the gates at the Facs

 

Good Luck!

 

Be careful!

GFP percentage on day 0 after transduction (day 3) may not reflect real percentage, because there is a delay in GFP expression. So, although the percentage may be low (or even zero), be patient. After a week on MS-5, most of the time it will have increased until steady levels. Take that value also as a day 0 value.