I would like to know how much volume of a blocking solution and the volume of antibodies solution works best for a cell suspension.
We resuspend the cells in 500 µL of blocking solution and then we add 100 µL of the antibodies solution (antibodies diluted in 100 µL of blocking solution) to a total volume of 600 µL. It looks like with some antibodies this is not working.
Do you use different incubation volumes? Do you adjust the cell concentration prior to blocking/incubating with primary antibody?
Thank you in advance.
Hello Álvaro Pascual
1. Use 100ul blocking solution. Incubate the cells on ice for 15-20 mins. Add 3-4 ml ice cold HBSS and centrifuge at 1500 rpm for 5 min at 4°C. Discard supernatant.
2. Resuspend the cell pellet in 100 µl primary labelled antibody. Incubate on ice for 15-30 mins in the dark.
3. After 15-30 mins incubation, add 3-4 ml ice cold HBSS and centrifuge at 1500 rpm for 5 min, 4 °C to remove unbound labelled antibody.
4. Wash the cells 2 times with centrifugation at 1500 rpm for 5 minutes each and resuspend the cells in 200 μl to 1ml of ice cold FACS buffer. Keep the cells in the dark on ice or at 4°C in a fridge until your scheduled time for analysis.
It would be better to discard the blocking solution before you add the primary labelled antibody solution.
Hope this helps.
Best.