Genekam MoAB Stainer: It is monoclonal antibodies Staining solution for MNC from the blood: To do the flow cytometry, you have to do the staining of cells from different sources e.g. blood, buffy coat, bone marrow aspirates with different monoclonal antibodies. We are offering you here a staining solution as it can deduce your error rate and give you better staining than your in house made. You are staining you MNC with different markers like CD34, CD19, CD20, CD4, CD3, CD133, CD105, CD90, CD45, CDCD8 and so on, you need this solution. This solution contains also RBC lysis solution also, hence you donot need to buy any RBC lysis solutions any more. You will finish this process within 20-25 minutes. It can be used with samples of all mammals.

This can be used for stem cells of human beings as well as other mammals e.g. rats, mouse, cattle, pig, dogs, horses, cats etc.

Genekam MoAB Stainer has two parts 1. erythrocyte lysis buffer, which is 10X solution and should be diluted to 1X in deionized water. 100 ml of 10X concentrate will make a quantity of 1X solution that is good enough to lyse a few hundred samples and 2. washing buffer. It needs your monoclonal antibody for staining.

Here are a few examples of MNC stainer to stain the cells for flowycytometry analyis with monoclonal antibodies, they can be done before starting the lysis of erythrocytes):

A. Lysis of erythroycytes of human peripheral blood or buffy coat:

1. Add 50 µl of blood to one flowcytometry tube
2. Add 150µ of Washing buffer
3. Add 15 µl of your conjugated monoclonal antibodies or only monoclonal antibodies. (it may vary according your need, please consult your monoclonal antibodies manufacturer)
4. Keep the above solution for 20 Minutes at room temperature in dark
5. Dilute the 10X RBC Lysis buffer to 1X working concentration with double distilled water or single distillted water. Warm the 1X solution to room temperature before use (recommended, but not necessary!)
6. Add 1.0 ml of 1X Genekam RBC lysis buffer to each tube containing up to 50 µl ofwhole blood and monoclonal bodies
7. Gently vortex each tube immediately after adding the lysing solution. Keep at room temperature, protected from light, for 7-10 minutes
8. Centrifuge 1600 rpm for 5 minutes. Aspirate or discard supernatant without disturbing pellet.
9. Resuspend the pellet in washing buffer and wash 2X while centrifuging for 1600 rmp for 5 minutes
10. Resuspend the pellet in washing buffer (e.g. 1ml, the volume of this step, you have to adjust according to your needs) and go for flow cytometry analysis. In stead of washing buffer, you can take your own buffer also.

B. Lysis of erythrocytes of human (mammal) bone marrow aspirate:

1. Add 50 µl of bone marrow aspirate to one flowcytometry tube
2. Add 150 µl of Washing buffer
3. Add 15 µl of your conjugated monoclonal antibodies or only monoclonal antibodies. (it may vary according your need, please consult your monoclonal antibodies manufacturer)
4. Keep the above solution for 20 Minutes at room temperature in dark
5. Dilute the Dilute the 10X Genekam RBC lysis buffer to 1X working concentration with double distilled water or single distillted water Warm the 1X solution to room temperature before use (recommended, but not necessary!)
6. Add 600 µl ml of 1X Genekam RBC lysis buffer to each tube containing up to 200 µl of bone marrow aspirate. (It is recommended to use 1:3 i.e. one part of bone marrow aspirate and two parts of Genekam RBC lysis. You may need to adjust the volume of RBC lysis buffer)
7. Gently vortex each tube immediately after adding the lysing solution. Keep at room temperature, protected from light, for 7-10 minutes
8. Centrifuge 1600 rpm for 5 minutes. Aspirate or discard supernatant without disturbing pellet
9. Resuspend the pellet in washing buffer and wash 2X while centrifuging for 1600 rmp for 5 minutes
10. Resuspend the pellet in washing buffer (e.g. 1ml, the volume of this step, you have to adjust according to your needs) and go for flow cytometry analysis. In stead of washing buffer, you can take your own buffer also.

C. Lysis of erythrocytes 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. Suspend the pellet
3. Add 50 µl of spleen suspension to one flowcytometry tube
4. Add 150 µl of Washing buffer
5. Add 15 µl of your conjugated monoclonal antibodies or only monoclonal antibodies. (it may vary according your need, please consult your monoclonal antibodies manufacturer)
6. Keep the above solution for 20 Minutes at room temperature in dark
7. Dilute the 10X Genekam RBC lysis to 1X working concentration with double or single distilled water. Now suspend the pellet in 5 ml of 1X Lysis solution
8. Incubate at room temperature for 7 minutes with occasional shaking
9. Add to Lysis Buffer with spleen cell with 20-30 ml of PBS buffer
10. Spin the cells (1600 rpm) for 5-7 minutes and discard the supernatant
11. Wash and centrifuge the cells 2 times at 1600 rpm for 5-7 minutes and discard the supernant. After that suspend your pellet in washing buffer (e.g. 1ml, the volume of this step, you have to adjust according to your needs) and go for flow cytometry analysis