Cell line transfection

HUDEP 2 nucleofection, culture and differentiation

Thawing of HUDEP-2

Preparation HUDEP-2 maintenance culture medium:

• In StemSpan SFEM (StemCell) lot: 15F64294, Exp 06/2017
• Doxicyclin “DOX” (1 ug/mL) from TAKARA BIO (actually bought to Sigma, stored at 4°C lab).
• SCF (100 ng/mL, stored at -20°C L2).
• EPO (3 IU/mL, stored at +4°C L2).
• Dexamethasone “DEX” (10E-6 M) from Sigma (stored at -20°C L2).
• 1% P/S (LifeTech ref:15140122).
• 1% L-glut (LifeTech ref: 25030024).

Thawing HUDEP-2 cells vial: 

- Quickly transfer vial from liquid nitrogen to 37°C water bath and pour cell suspension in SVF.

- Spin at 300g for 5 minutes.

- Resuspend pellet in fresh HUDEP-2 maintenance medium. 500µL.

- Count

- Resuspend at 10⁶/mL

Following days: plated at 500,000/mL, except over the weekend at 300 000/mL

Nucleofection

(Kit Amaxa Cell Line Nucleofector Kit - VCA-1003) - Nucleofector I, program L-29 for HUDEP-2 cells

Everything is done under the hood.

9 samples, 7 gRNAs to test, 2 in duplicates

• Prepare 24 well plates containing 1mL HUDEP-2 medium per well (according to the number of samples).
• Prepare IMDM + 20% FBS medium (5mL per condition) and keep it warm => 50 mL total = 40 mL + 10 mL FBS

Prepare Amaxa solution / per sample (100µL) = 82 µL Nucleofector solution V + 18 µL Lonza Supplement I solution

For all samples (9) = 738µL V and 162µL Supplement I

• Prepare ½ dilutions of gRNAs: 5µL + 5µL of sterile TE
• Prepare plasmid mix (in red: dilution that was used) and resuspend in 95uL Amaxa solution.

• Counting of HUDEP-2
• Pipette 1E6 HUDEP-2 cells in 1,5mL tubes and centrifuge at 300g 5min.

The next steps are done ++ quickly and 1 sample at a time.

• Prepare the pipette + cuvette + pipet set at 110µL
• Resuspend cell pellet in 100uL nucleofection solution + plasmid mix and transfer in electroporation cuvette + tap the cuvette to homogenize.
• Quickly set up L-29 program on Lonza electroporator machine and perform electroporation by pressing button OK.
• Quickly pour in the cuvette around 500uL recovery medium (IMDM + 20% FBS) with plastic manual pipet.
• Take the cell suspension from the cuvette (2 times with 500 µL buffer) and pour in 5mL recovery medium.
• Centrifuge all cell samples together at 300g for 5min.
• Remove supernatant to keep around 100uL recovery medium in the tube.
• Gently resuspend the pellet.
• Unscrew the lid of the tube to let CO2 enter in it and keep cells at 37°C and 5% CO2 for 15min.
• Pour the 100µL of cells in the 1mL of HUDEP-2 warm medium in the 24 wells plate.

1 day after nucleofection – GFP expression / sorting 

• Cells are recovered (the whole culture, without counting). They are already in 1 mL, take off 400µL of SN.
• Filter with blue filters (spin the tip around the filter to avoid clusters).
• Keep on ice.
• Prepare FACS tubes with 500µL of FBS 100% - keep on ice.

Sorting with Sony SH-800

• In experiment template, search for the last experiment (HBG prom…) Open. 
• FSC: tick height and width
  BSC = SSC: tick height
  FL1: tick area.
  Laser: GPF= 488nm (emission wavelength =blue), we can untick the 561 nm laser.
• Are you sure: yes
• Laser 488 nm turned on = blue square
• Active experiment: rename and Create a new experiment.
• We can change the name of the tubes in the acquisition dashboard. 
• It’s saving automatically.
• Assign the sorting gate: Gate GFP+ = “F”, down arrow and R button ticked on the right in the Sort CTL panel. (Sort statistics will show up only on the right side when sample is running).
• Check that the drop are stable in the droplet viewer: the green button should not be flashing.
• Change the tube holders: for the sample containing the cells (to be sorted) = single tube holder, small one for FACS tubes. For the sorted cells: double tube holder, also small and transparent. Place it well + strongly.
• Open left door
• Msg = “ok”
• Vortex the 1^st sample with cells and containing tube with FBS and place them.
• Put it on the right side and place collection tube on the left side, under the right side – because sort = R.
• Press load collection
• Sample pressure = 7
• Tick autorecord = it will record 100,000 cells automatically.
• Press START= start aspiring and displaying the cells after few sec.
• Press Sort and Record Start
• Sample pressure = 10.
• “recording has been stopped by meeting the stop condition” = ok, end of the recording.
• When the sample is almost empty: “Stop”. Otherwise: “end of the sample detected”, stops automatically but aspire some air.
• Take back the sorted population, and tap with finger to homogenize the FACS flow and FBS.
• Next tube, load collection, sample pressure 7, autorecord, Start, Sort and record start, sample pressure 10, Stop.
• =9 min per sample
• Save experiment: right click on the name of the experiment (above all the tubes), export FSC file, parameter by default, select folder on the server under “ordinateur”, export : ok.

Count the cells in the L2 and plate at 500 000/mL.

Every day after the sorting= count the cells, change the medium and plate at 500 000/mL, except over weekends at 300 000/mL.

Day 0 differentiation

Cell counting – 1 mL 1/10^th 

FACS: eCr and iCr HbF

eCr: NM, CD36 V450, CD71 FITC, GPA PE-Cy7 mono + Mix

iCr: NM perm, GPA PE-Cy7 perm, HbF APC perm + Mix

FACS: eCr:

• GPA PE Cy7 (1/10^th): 5µL 
• CD71 FITC: 5µL
• CD36 V450: 2,5µL

iCr: 

• GPA PE Cy7 (1/10^th): 5µL
• 15 min 4°C
• Wash PBS 1% FBS
• Vortex
• 250µL Cytofix Cytoperm
• 20 min 4°C
• 1 mL perm/wash – spin
• 100µL 1X perm wash buffer containing 1µL HbF APC (make a mix)
• 30 min 4°C
• Wash perm wash
• Resuspend in 300µL PBS 1% FBS

Day 2

Cell counting – plating at 500 000/mL 

Day 5

Cell counting – plating at 500 000/mL 

Day 7

Cell counting – plating at 500 000/mL 

Day 9

 Cell counting

Analysis

HPLC Single chains

500 000 cells resuspended in 40µL, 35µL injected.

HPLC tetramers

0.5 M cells lysed in 200 µL

50,000 cells injected (20 µL of lysate + 20µL H2O)