Jan 09, 2026
Protocol to generate stable cell line using HEK293 Flp-ln TREx cells
- Julia Heiby1
- 1FLI Leibniz Institute on Aging
Protocol Citation: Julia Heiby 2026. Protocol to generate stable cell line using HEK293 Flp-ln TREx cells. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk8yk1l5r/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 21, 2024
Last Modified: January 09, 2026
Protocol Integer ID: 106097
Keywords: hek293 flpin trex cell, ln trex cells transfection, stable cell line, using hek293 flp, cell, hek293 flp
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Abstract
Transfection of HEK293 FlpIn TREx cells, to generate stable cell lines.
Materials
- Centifuge 5810R (#5811000015, Eppendorf)
- Safety cabinet (Thermo, #Safe S2020 1.2)
- Automated cell counter (Biorad, #TC20)
- Trypan blue solution for cell counting (Sigma, #T8154)
- Counting Slides for cells (Biorad, #145-0011)
- Incubator (Thermo, #BBD 6220, CO2 Incubator), settings: 5% CO2, 95%rH, 37°C
- Milli-Q water system (Merck, Advantage A10)
- Flp-In T-REx 293 Cell Line (RRID: CVCL_U427)
- XtremeGENE9 A Transfection Reagent (Roche, #6365779001)
- 6-well plate (Falcon,# 353046)
- 15ml and 50ml Falcon (Corning, #352096 and #352070, respectively)
- 1.5ml tubes (Eppendorf, #0030 120.086)
- Serologicla Pipettes (Greiner, cellstar)
- Opti-MEM (Gibco, #31985-047)
- pOG44 Flp-Recombinase Expression Vector (Invitrogen, #V600520)
- Blasticidin (Gibco, #R210-01)
- HygromycinB (Invitrogen, #10687010)
- Trypsin-EDTA (0,05%, Gibco, #25300-062)
- DMEM high glucose (4,5g/L) (Sigma, #D6429)
- FBS, fetal bovine serum (Sigma,#F7524)
- DMSO, dimethyl sulfoxide (Sigma, #D2438-5)
- Penicillin/Streptomycin (Gibco, #15140-122)
Troubleshooting
Day 1
Seed 200 000 on a 6-well (at this point use medium with no antibiotics at all)
Do not forget to seed a well for no-DNA control transfection.
Day 2
Transfect cells on a 6-well plate with XtremeGENE9 from Roche (XTG9-RO), following instructions of the manufacturer. Pre-Mix under the hood: Use 1:9 of plasmid : pOG44
- 100µL Opti-MEM (or DMEM with no FBS, no antibiotics)
+ 3µL X-tremeGENE9 Transfection medium
+ 1µl of Plasmid DNA (100ng; containing target sequence to transfect)
+ 4.3µl pOG44 (900ng; pOG44 Flp-Recombinase Expression Vector from Invitrogen)
Incubate 15 min at RT (change to medium with no antibiotics if required)
Add dropwise to wells
Distribute evenly by gently moving flask
Day 3
Trypsinize and transfer cells onto a p150 plate (25ml medium)
Day 4
Add the corresponding antibiotics for selection (usually, Blasticidin + HygromycinB. For 20 mL of medium: 30 µL Blasticidine + 40 µL HygromycinB (from stocks 10 mg/mL Blasticidine and 50 mg/mL HygromycinB).
Following days / weeks
- Replace medium with fresh antibiotics twice a week until colonies are seen in your transfection but not in the negative control (no-DNA).
- When colonies start being too big, trypsinize them onto the same dish to separate the cells and allow them to grow better. The day after trypsinizing them, add fresh antibiotics.
Final antibiotics concentrations:
Blasticidin: 15 µg/mL
HygromycinB: 100 µg/mL
This usually takes 2-3 weeks for HEK Flpln TREx cells.
Advice
For more information see info given by Invitrogen or Roche
Preparing cryo-cultures
Buffers and reagents
growth medium – pre-warmed to 37°C: (stored in the fridge)
DMEM high glucose (4,5g/L) -500ml
10% FBS (heat inactivated for 30min at 56°C) -50ml
+ antibiotics depending of the cell type
freezing medium (4°C):
FBS (heat inactivated) -45ml
10% DMSO -5ml
PBS – pre-warmed to 37°C (without calcium and magnesium/ stored in the fridge)
0,05% Trypsin-EDTA (1x)-pre-warmed to RT (contains 0,05% phenol red
as pH indicator, keep it only short time outside the fridge because of
autodigestion- stored in the fridge)
- grow cells to a density of 80% confluence in growth media with antibiotics (1 day)
- wash the cells one time with 5ml PBS
- add 0,05% Trypsin-EDTA and incubate at 37°C
- stop trypsinization by adding of growth media, transfer cell suspension to a conical tube
- pellet cells at 500xg for 5min at 20°C
- aspirate the supernatant and resuspend the pellets in 4°C freezing medium (at about 1 to 4x106 cells/ml)
- dispense 0.5ml up to 1ml aliquots into labeled vials and place them in a cell freezing container
- place freezing container into -80°C freezer o/n
- Transfer vials in liquid nitrogen for long term storage
Protocol references
Following the Manufacturers instruction for the X-tremeGENE‱ 9 DNA Transfection Kit from Roche.