Hello Kenneth Ng,

What I would recommand is first to check your cells regularly for mycoplasma using PCR test, and to try to have a sterile culture.

In order to concentrate your lentivirus, you can follow the protocol from Didier Trono at EPFL: https://www.epfl.ch/labs/tronolab/

Here is the extract from his protocol regarding the supernatant containing your lentivirus:

1. Centrifuge 5 min at 500 × g, 4°C, to pellet detached cells and debris.

2. Filter the supernatant using a 0.22-μm filter unit.

3. Pipet the supernatant into clear ultracentrifuge tubes. Ultracentrifuge 120 min at 50,000 × g, 16°C. Gently discard the supernatant by inversion. Let the tube dry inverted.

After centrifugation, do not let the pellet dry too much, around 2-3 min is sufficient, then remove the remaining medium on the tube with a paper .

4. Resuspend the pellet (not always visible) with PBS1X, by pipetting first 10-15 times all around pellet, and then up and down 15 times. The vector pellet of one tube can be resuspended in a minimal volume of 30 μl. In this case, a 1000-fold concentration will be obtained. As there is always some medium remaining even after drying, add first only 15μl of PBS1X per tube. The advantage of this remaining medium is that it brings some proteins important for viral particles stabilization. Practically, had 15μl to each ultraclear tube and an extra 15μl to the first one. After resuspension of the first tube, recover 15μl in a sterile eppendorf and transfer the remaining volume to the second ultraclear tube Proceed sequentially until the last tube. Try to avoid bubbles when resuspending the pellet since their presence will result in decrease of final volume, and hence decrease of yield.

5. Clear the final concentrate by a brief centrifugation (around 5 seconds) at maximum speed on a bench top centrifuge. Aliquot the supernatant and store at 80°C.

What you can do in the end is to titrate your virus using lentiviral titrattion kit.

All the best,

Maxime.