Author: Nora

Transfection of 293T cell with CaCl2 for production of lentivirus (2^nd generation)

Materiel

• ProFection Mammalian Transfection System (Calcium Phosphate), cat: E100
• Polybrene
• Puromycin

Protocol

• Day before: Seed 5-6 million cells in a T-75 flask the day before transfection. Leave a tube of media in the incubator overnight with the cap loosened.
• Day 1: change the media of the cells.
• Day 1 after 4 hours: prepare the transfection mix in 1.5 vial as follows:
  • Plasmid of interest (6.7ug): x uL
  • 91 (6.7 ug): y uL
  • VSVG (6.7 ug): z uL
  • Water (up to 438 uL): w ul
  • 2M CacL2: 62 uL
• Then transfer 500uL of 2x HBS to a 15mL conical tube. Add the plasmid mix drop by drop into the 2xHBS while vortexing. Leave at room temperature for 30min.
• Add the mix to the cells drop by drop while swirling the flasks.
• Day 2: Next evening, remove the media with the transfection mix, rinse once with PBS and add media specific to the target cells (ex: DMEM+FBS+PS for SH-SY5Y cells). Note: the media might already contain lentiviral vector, but it is usually discarded.
• Day3: Next morning, collect the media with transfection mix, filter through a 0.2 um PVDF filter and add polybrene to a final concentration of 8ug/ml. Add this virus mix to the target cell flask (i.e: SH-SY5Y cells in T25 flasks).
• Replenish the 293T cells with fresh 5mL cell media (like step 6-) and repeat the harvesting and infection procedure at the end of the evening (like step 7-).
• Day 4: Repeat as step 7-. Note: 297T cells might have been dislodged from the flask, then do not repeat.
• Start antibiotic resistance (puromycin at 5ug/ml but puromycin concentration must be determined for the cell type) 2-3 days after transduction of the target cells (ex: SH-SY5Y) and for 5 days.

Note:

• 297T cells has tendency to detach from the flask
• Do the step 3 when cells are at a confluence of about 60%, similar for the target cells at step 7.
• Add the smallest amount of medium to cover the cells while keeping it concentrated with virus. We transfer the whole medium (no centrifuge step) so you don’t want to have to much medium and you want to make sure your lentiviral vector will transduce the wanted cells, example: 3.5mL for a T25 flasks.

Western Blot

Preparing the sample:

• Prepare aliquot of Laemmli with 2-mercaptoethanol (cat. 161-0710, bio-rad) together.
  • If 2x Laemmli (cat. 161-0737, bio-rad) sample buffer: add 50uL of 2-mercaptoethanol to 950uL of Laemmli
  • If 4x Laemmli sample buffer (cat. 161-0747, bio-rad): add 100uL of 2-mercaptoethanol to 900uL of Laemmli
• Mix the Lammli, protein and HBSS to reach the protein mass and total volume wanted per well. See the excel template. (I am using 20ug of protein per well)
• Warm the sample at 90C for 5 min on a heat block.

Electrophoresis:

• Prepare 1L of running gel 10x SDS-PAGE (250 mM Tris, 1.92 M glycine, 1% SDS, pH8.3)
  • Tris base (trizma): 30.30g
  • Glycine: 144.10g
  • SDS: 10g
  • diH2O/Milli-Q Water to 1L
  • Or buy already made 10x Tris/Glycine/SDS (cat: 1610732, bio-rad)
• Prepare 1L of running gel at 1%
  • 100mL of 10x SDS-PAGE
  • diH2O/ Milli-Q Water to 1L
• Open the pre-made gel packaging (possibility to make the gel yourself). I use 4-20% MP TGX Gel (cat: 4561096, cat: 4561093), remove the green tape at the bottom, place the gel in the holder and then the holder inside the tank, and finally remove the comb. If using only one or three gel, use the dam insert. Use only one insert if you use 1 or 2 gels.
• Fill the middle chamber of the insert(s) and the tank with running buffer. Straight up any well wall that has collapsed with a pipette tip. Use a paster pipette to rinse the gel holes with the running buffer from the tank to remove acrylamide debris.
• Load the wells with the protein ladder (exemple: Precision Plus Protein Dual Color Standards, cat. 1610374) and your samples.
• Run the gel at 200V until the sample dye front reach the bottom of the cassette. (If the equipment doesn’t detect input, fill the middle chamber of running gel as sometimes it doesn’t detect it if not enough liquid.)

Dry transfer:

• When the gel has finished to run. Remove the cassette and open to take the gel out.
• Transfer the gel inside the dry transfer membrane. We use Trans-Blot Turbo Mini 0.2um PVDF Transfer Packs (cat: #1704156) and the gel will be sandwiched between a bottom and top part.
• Put the whole thing inside the Trans-Blot Turbo Transfer System and select the appropriate transfer time.

Blocking:

• Prepare 1L of 10% TBS (Tris: 200mM, NaCL: 1500 mM)
  • 24g of Tris Base
  • 88g of NaCL
  • 900mL Milli-Q water
  • Adjust pH to 7.6 with HCL
  • Adjust final volume to 1l with Milli-Q Water
  • Or buy already made 10% TBS (cat: J60764.K2, thermo scientific)
• Prepare 1L of 1% TBS
  • 100mL of 10x SDS-PAGE
  • diH2O/Milli-Q Water to 1L
• Prepare 1L of 1% TBS, 0.1% Tween20 (TBST)
  • 100 mL of 10X TBS Stock Solution
  • 900 mL of Milli-Q® Water
  • 1 mL of Tween® 20 detergent
• When the transfer is done, take the membrane and deep it into a blocking agent for 1h in a rocking shaker. Use 5% milk powder or 5% Bovine Serum Album powder (especially for phosphorylated protein) in TBST. (Use TBS if doing fluorescent western-blot!)
• Rinse membrane in 1x TBST for 5 minutes

Primary Antibody

• Prepare first antibody dilution in 5mL 1% blocking agent in a 50mL falcon tube (optimise the primary antibody dilution, if 1:1000, use 5uL antibody) and transfer the membrane in the 50mL falcon tube for 1h at room temperature or overnight at 4C (or like colleagues: overnight at room temperature) in a gentle constant rocking (level 6 in our lab rocker)
• Wash the membrane with 1% TBST three time for 10 minute each on the rocking shaker

Secondary Antibody

• Prepare secondary antibody dilution in 5mL 1% blocking agent in a 50mL falcon tube (optimise the secondary antibody dilution, if 1:5000, use 1uL antibody) and transfer the membrane inside the falcon tube for 1h room temperature in a gentle constant rocking (level 6 in our lab rocker)
• Wash the membrane with 1% TBST three time for 10 minute each on the rocking shaker

Detection method:

1. Chemiluminescence HPR substrate detection

• Use plastic transparent hot plate cover to lay out enough plastic cling film to later being able to wrap the whole membrane. Wet the hot plate cover before putting the cling film to make it easier to work with cling film. Use a rolling pin to lay out well the cling film is necessary.
• Mix ECL kit (Immobilon Western Chemiluminescent HRP Substrate, cat: WBKLS0500) to have 5mL volume per membrane
• Place the membrane on top of the cling film and right away put the 5mL of ECL substrate on the membrane. (You don’t want the membrane to dry out). Incubate for 1 to 5 minutes at room temperature. Then drain the excess water with blue roll paper. Cover the blot with the cling film and expose it for detection.

2. Fluorescence detection

• Let the membrane dry
• Use the Chemodeck to picture the membrane

From RNA extraction to qPCR via cDNA and primer testing

We are now using the Mic qPCR Cycler instead of RotorGene, however, the protocol is the same, just use the Mic (and Mic tubes) instead.

Cell pellet collection

• Remove medium
• Wash the cell with PBS or HBSS
• Remove the medium
• Collect the cell using either method
  • Scrapping method:
    • Add 1ml of HBSS or PBS
    • Scrap the cell and collect in an Eppendorf (1.5ml Eppendorf should be enough)
  • Trypsin method:
    • Add 2ml of trypsin, then inside the incubator for one to two minutes.
    • Neutralise the trypsin using 8ml of DMEM + FBS + PS (or any trypsin neutralising agent of your choice).
    • Transfer all into a 15ml falcon tube
  • Centrifuge and remove the supernatant.

RNA extraction and quantification

• RNA extraction following manufacturer instruction of either:
  • Monarch Total RNA miniprep Kit
  • Direct-zol™ RNA MiniPrep Plus
• Quantification using 1.5uL on Nanodrop. Read concentration in ng/ul and the A260/A280. For Monarch kit I constently have a A260/A280 between 2.01 to 2.08. For Direct-zol kit I have a A260/A280 between 1.80 – 1.90.

Making cDNA from RNA

• Following the manufacturer protocol of High Capacity cDNA Reverse Transcription Kit, using Kit without RNase inhibitor, and using 2ug of RNA. Total volume will be 20uL.
  • If 2ug is not possible use 1ug.
• After the cycler (per manufacturer protocol) is done or before processing the sample, add 180uL of DNAse/RNAse free water to make all samples at 2ug/200ul = 10ng/ul.
  • If using 1ug, add 80ul to make all samples at 1ug/100ul = 10ng/ul.

Testing primers

• Factor dilution of 3, cascade dilution of cDNA:
  • Stock cDNA at 10ng/ul
  • Take 66.7uL of the stock cDNA into 133.3ul of DNAse/RNAse free water (total volume = 200ul) = 3.33ng/ul
  • Take 66.7ul of the cDNA above into 133.3ul of DNAse/RNAse free water = 1.11ng/ul
  • Take 66.7ul of the cDNA above into 133.3ul of DNAse/RNAse free water = 0.37ng/ul
  • Take 66.7ul of the cDNA above into 133.3ul of DNAse/RNAse free water = 0.12ng/ul

I respectively name the tubes 1x cDNA (for the stock cDNA), 3x cDNA, 9x cDNA, 27x cDNA, 81x cDNA for each successive dilution. Name them according to your wish.

• qPCR to test the primer sets per well:
  • 10uL of PerfeCTA SYBER green Super Mix
  • 1 uL Forward primer (at 10uM)
  • 1 uL Reverse primer (at 10uM)
  • 8 ul cDNA. Need 5 Rotor Gene Q Strip Tubes. Each strip contains 4 wells for one dilution of cDNA in triplicate and one control NTC using DNAse/RNAse free water. 5 strip tubes for each cDNA dilutions (10ul of 1x = 100ng, 10ul of 3x = 33.3ug, 10ul of 9x = 11.1ng, 10ul of 27x = 3.7ug, 10ul of 81x = 1.2ng).
• Run qPCR according to the manufacturer: 3 steps.

qPCR

• PerfeCTa SYBR Green SuperMix and a primer set diluted at 10uM make a master mix following this recipe per sample (always add for extra samples to compensate pipetting lose):
  • 10uL of PerfeCTA SYBER green Super Mix
  • 1 uL Forward primer (at 10uM)
  • 1 uL Reverse primer (at 10uM)
  • 3 ul DNAse/RNAse free water

I use Rotor Gene Q Strip Tubes and Caps 0.1ml, each strip tubes is 4 wells, ideal for triplicate + ntc per sample.

• Pipette 15uL in each well (triplicate + ntc per sample)
• Pipette 5ul of cDNA ( = 50ng) in 3 wells (triplicate) and 5ul of DNAse/RNAse free water in one well (ntc).
• Run qPCR according to the manufacturer: 2 steps.

BCA protein assay protocol

1. Protein extraction from cell culture
   • Rinse the cells with DPBS
   • Add 5 mL of DPBS and scrap the cells
   • Centrifuge 10 min at 1000g
   • Remove supernatant and store the cell pellet in -80 freezer until needed.

2. Lysis for protein extraction
   • Mix 350 uL of ice-cold Pierce IP Lysis Buffer (cat. 87787, Thermo Fisher) + 3.5uL of Halt Protease and Phosphatase Inhibitor Cocktail (cat. 78440, Thermo Fisher) = 353.5uL total volume
   • Resuspend the cells with the ice-cold Pierce lysis buffer mix and incubate 5 min at room temperature with periodic mixing
   • Centrifuge at 13000g for 10m at 4C.
   • Transfer the supernatant to a QIAshredder homogenizer column (Cat. 79656, Qiagen) and centrifuge one minute (max 700uL and 2min centrifugation per load). Store in -80 Freezer until needed. (Tips: Make a small aliquot of 20uL for BCA assay below, it will avoid you to defrost the whole sample)

3. Protein concentration assessment using the kit Pierce BCA Protein Assay Kit (Cat. 23225, Thermo Fisher)
   • Make aliquot of diluted albumin standard ampules (included inside the kit: Albumin Standard Ampules, 2mg/mL, 10 x 1 mL) in HBSS to reach the dilution rage of 50; 70; 100; 300; 500; 700; 1000 ug/mL (you can adapt the range based on your cell types/quantity of protein expected by swapping 50 for 2000 ug/mL).
   • Transfer 20uL of protein in an Eppendorf tube and add 130uL HBSS, total volume = 150uL
   • Similarly, with the protein concentration range, transfer 20ul of each dilution range in different Eppendorf and add 130uL of HBSS on each tube to have a total volume of 150uL in each tube.
   • For the negative control, put 150uL HBSS in an Eppendorf.
   • Mix reagent A and B (ratio 50:1, Reagent A:B) to have 850uL per sample. I do 850ul A + 17uL B = total volume of 867uL to have a little extra. (1 negative control + 7 protein dilution ranges + N samples)
   • Add in each prepared Eppendorf 850uL of A+B mix, total volume 1mL.
   • Incubate all Eppendorf tubes for 30 minutes at 37C (for range of 20-2000ug/mL). Can also do 2h at room temperature (for range of 20-2000ug/mL) or 30 minutes at 60C for range of 5-250ug/mL). Tubes will progressively change colour based on the protein concentration.
   • Transfer 300uL per sample per well (triplicate) in a 96 well plate and read absorbance at 562nm in a plate reader.

4. Follow the Excel sheet for the assessment of protein concentration.