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When using the RAFT Culture System for embedding cells and having a layer of other cell type on top of it (example a cellular barrier model), is it possible to remove the top layer of cells later?

If you have a layer of cells imbedded within the collagen matrix and a second cell type plated on top of the collagen matrix, it should be possible to detach the top layer of cells using trypsin, which will keep the collagen scaffold intact. I would...

Do we do customs of fresh bone marrow in different media?

We can ship fresh bone marrow in any media that the customer requests as long as the send the media to use beforehand. Any changes to the process at the donation facility are not permitted due to the IRB. So you cannot change the amount of heparin or...

How does Lonza perform the cell count on the fresh, unprocessed bone marrow? Does Lonza guarantee a minimum viability for the fresh, unprocessed bone marrow?

Lonza use a Coulter Counter to perform the total cell count on the fresh, unprocessed bone marrow. Lonza does not perform viability testing on this product, but when shipped overnight, viability is typically around 80%.

Which program should I use for co-transfection of RNP and CRISPR/Cas9 complexes ? The paper "Optimized RNP transfection for highly efficient CRISPR/Cas9-mediated gene knockout in primary T cells from Aikiko Seki and Sascha Rutz showed different programs as you have in the Lonza optimized Nucleofector Protocols?

Their publication in the Journal of Experimental Medicine is an advanced optimization for gene knock out in primary human and mouse T cells. The final protocol achieved 85-98% gene knock out for several tested receptors.The group has tested and...

How would you further cultivate the cells after the formation of a RAFT Cellular structure?

For the 96 and 24 well RAFT™ Kit format, you simply put the media on top of the RAFT structure laying already in the well, and cultivate the cells, as you classically do in 2 dimensions. For high cell number in RAFT™ Cultures you might use twice more...

How long does it take for the fresh bone marrow sample to arrive from the moment of harvest?

Barring any delays at airport customs, and assuming there are no weather related delays, the sample can be delivered anywhere in the continental United States within 26 hours of collection (same day delivery may be available for local deliveries,...

When a RAFT 3D Culture is subjected to an immunofluorescence labeling, should one expect a high background coming from antibody cross reactivity with the matrix?

Our Technical Note “Immunofluorescence Visualization of Fibroblast or Cancer Cells in RAFT™ 3D Cell Cultures” shows no immunofluorescence cross reactivity between the used antibodies and the rat collagen matrix, even with secondary antibodies...

Is there a media recommendation for differentiating CD34+ cells into erythrocytes ?

Lonza offer a differentiation kit for erythrocyte differentiation of CD34+ cells (Lonza catalog no. PT-4514 and PT-4515).

Which Nucleofector Protocol should I use for genome editing and transfection of CRISPR/Cas9 into my cells?

Please use always the ready-to-use protocol for the specific cell type. The optimized programs are cell type specific and do work for any kind of substrates. You can transfect small and large DNA vectors, mRNA, siRNA, gRNA, peptides and proteins with...

What are the size of CD34+ Cells?

When resting, they are about 8-10 microns.
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