In order to do an ELISPOT, I have to isolate mouse splenocytes from mice which I vaccinated. Every time, I did this isolation, by following the common protocol used in my laboratory, two or three of my splenocytes suspension begin to die and form a lysate into the tube while I was counting the living cells before using them. In these suspensions, a cluster of cells or cellular debris appeared on the suspension (a white mass floating in the suspension) and the number of cells living decreased. The others suspension remain unchanged until the end of the preparation and nobody in the lab have had this problem before.
Does anyone has had this problem before and found a solution in order to avoid that this happen again?
PS : This is the procedure I followed :
- Cut out the spleen and place it into an eppendorf of RPMI medium (+Peni/Strepto + beta-mercaptoethanol) on ice
- Place the spleen into the cell strainer. Using the plunger end of the syringe, mash the spleen through the cell strainer into the tube an universal tube (falcon tube)
- Rinse the cell strainer with 5mL RPMI medium (+P/S + beta-m). Discard the strainer
- Centrifugate at 300xg during 5-10 minutes at room temperature
- Discard supernatant and resuspend pellet in 1mL ACK lysis buffer per spleen (0.15M NH4Cl, 1 mM KHCO3, EDTA 0.1 mM). Incubate at RT for 30 seconds to 1 minute. Add 5mL RPMI (+P/S + beta-m) and spin as before.
- Discard supernatant and resuspend pellet in 1mL RPMI (+P/S + beta-m + 2% mice normal serum) per spleen, discarding any dead cell mass (which appeared normally at this step)
- Count cells (dilute 10μL cell suspension in trypan blue, and count with hemacytometer).