Genekam Biotechnology AG has developed a new method to isolate the mononuclear cells (MNC) from different sources like bone marrow aspirates, buffy coats, blood, umbilical cord blood and other sources. Usually one uses density gradient centrifugation, which is a time consuming and need a lot of precautions during the pipetting of fluids on density gradient. Making a mistake means that you are going to lose the whole samples. Density gradient methods are not good for isolation of MNC from the sources, where you have less than one ml volume and more you get only a part of MNC as you cannot take out the cells from white ring. Genekam Biotechnology AG has developed one method, where you can isolate all cells during your isolation available in sample within 20 minutes. Not only this method is faster, but it is also economical and let you isolate the MNC from as little as 25µl of samples along with the larger size samples. This is a sterile product.

Genekam Quick MNC isolator is ready to use sterile solution for isolating the mononuclear cells (MNC) quickly from blood, buffy coats, bone marrow aspirates, spleen etc of different mammals like human, rat, primates, cattle, sheep, goat, pig, horse, camel, dog, cat etc.

Here are a few examples of application of Quick MNC isolater:

A. Isolation of MNC from human peripheral blood or buffy coat:

1. Add 900µl of Genekam Quick MNC isolater to each tube containing up to 100 µl of whole blood or buffy coat in one sterile tube as this is ration (1:5) of solution to the sample e.g. if you want to isolate MNC from 2ml of buffy coat and add 8 ml of Quick MNC isolator. You have to decide howmuch blood or buffy coat sample do you want to use to isolate the MNC as you will get a lot of MNC against the conventional method of density gradient centrifugation.
   Warning: Kindly work with smallest volume of sample to gain some experience with this kit instead of destroying whole sample for first time.
2. Gently vortex each tube immediately after adding the solution. Keep at room temperature, protected from light, for 7-10 minutes.
3. Centrifuge 1600 rpm for 5 minutes. Aspirate or discard supernatant without disturbing pellet.
4. Resuspend the pellet in the appropriate buffer (usually it is PBS in most of applications) and wash 2X while centrifuging for 1600 rmp with brake for 5 minutes each.
5. Resuspend the pellet and proceed with further procedures e.g. counting the number of MNC and go for your analysis e.g. cell culturing.

B. Isolation of MNC of human bone marrow aspirate:

1. Add 400µl of Genekam Quick MNC isolater to each tube containing up to 200µl of human bone marrow in one sterile tube as this is ration of solution to the sample (1:3 ration) e.g. if you want to isolate MNC from 2 ml of bone marrow aspirate and add 4 ml of Quick MNC isolator. You have to decide about the volume of bone marrow you want to use to isolate the MNC because you will get a lot of MNC against the conventional method of density gradient centrifugation.
   Warning: If you do not have any experience with this kit, kindly use smallest volume of sample to have experience with this kit before you destroyed the whole sample as you may have to adjust ration e.g. you have still erythrocytes at the end, you have to use more MNC isolator (1 volume sample and 4 volume MNC isolator e.g. 200 µl sample and 600 µl of MNC isolator) or wash one time more. Usually the above said protocol is good enough to isolate MNC.
2. Gently vortex each tube immediately after adding the MNC isolator solution. Keep at room temperature, protected from light, for 7-10 minutes.
3. Centrifuge 1600 rpm for 5 minutes. Aspirate or discard supernatant without disturbing pellet.
4. Resuspend the pellet in the appropriate buffer (usually it is PBS in most of applications) and wash 2X while centrifuging for 1600 rmp with brake for 5 minutes each.
5. Resuspend the pellet and proceed with further procedures e.g. counting the number of MNC and go for your analysis e.g. cell culturing.

C. Isolation of MSC from of mouse spleen:

1. Prepare a single cell suspension after harvesting the mouse spleen.
2. Pellet the cells by centrifugation (1600 rpm); discard the supernatant.
3. Now suspend the pellet in 5 ml of MNC isolator isolation.
4. Incubate at room temperature for 7 minutes with occasional shaking.
5. Add to MSC isolator solution with spleen cell with 20-30 ml of PBS buffer.
6. Spin the cells (1600 rpm) for 5-7 minutes and discard the supernatant.
7. Resuspend the pellet in the appropriate buffer (usually it is PBS in most of applications) and wash 2X while centrifuging for 1600 rmp with brake for 5 minutes each.
8. Resuspend the pellet and proceed with further procedures e.g. counting the number of MNC and go for your analysis e.g. cell culturing.