To know about the general procedure or flow cytometry, it is suggested to learn and follow the following procedure:
Harvest and clean the cells and alter cell suspension to a concentration of 1-5 x 106 cells/mL in ice-cold PBS, 10% FCS, 1% sodium azide. The fact is that cells are typically stained in polystyrene round bottom 12 x 75 mm2 Falcon tubes. But they may be stained in any box for that you have the appropriate centrifuge e.g. Eppendorf tubes, test tube, and 96-well round-bottomed microtiter plates. It should be noted that cells must be centrifuged completely so the supernatant fluid may be removed with little lack of cells, however not so hard that the cells are daunting to resuspend.
While doing this step during the procedure of FACS Protocol, it is recommended staining with ice cold reagents and at 4°C, as low temperature and presence of sodium azide save you the modulation and internalization of surface antigens. Moreover, internalization can result in a loss of fluorescence intensity.
Put 0.1-10 μg/mL of conjugated primary antibody. Moreover, dilutions are done, if important, must be made in 3% BSA/PBS. Now incubate for half an hour in dar at room temperature or 4°C. This step would need optimization.
Clean the cells 3 x by centrifugation at 400 g for five min and resuspend them in 500 µL to 1 mL of ice-cold PBS, 10% FCS,1% sodium azide. Now secure the cells in the darkish on ice or at 4°C in a fridge till your scheduled time for evaluation.
For effective effects, analyze the cells on the flow cytometer as quickly as feasible.
Moreover, for the best results, it is suggested to indulge in analysis on a similar day. In a case of extended storage (16 hours) and for higher flexibility in planning time on the cytometer, it is suggested to resuspend cells in 1% paraformaldehyde to save from deterioration.
While FACS Staining Protocol procedure, if you require to holding more than an hour, then you have to fix the cells once you have finished step 3. Moreover, if so, you can preserve these for several days. Furthermore, this would stabilize the light scatter and inactivate most biohazardous agents.
Controls will require fixation using the identical system. Cells should not be constant if they require to remaining viable. There are numerous strategies to be had, please consult with the fixation phase inside the indirect staining protocol. The fixation for special antigens will require optimization by means of the user.
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