Dear All
I am collection blood from Balb/c mice through tail vein (a tumour bearing mice). Once blood flow through the heparin microhematocrit capillary and reach the 3/4 I breath hard on to EDTA coated (BD) tube shake it to prevent clotting. staining procedure has been conducted immediately or at least in 30 minutes after collection! stainings are performed in 2%FBS in PBS (PBS pH is 7.2 or 7.4! and prepared fresh before start!)
I am using biolegend 10X RBC lysis buffer. to make it 1X, In 1 experimen diluted it with Millipore water. Added 1mL into the tube and incubated on ICE and RT on 2 separate experiments at the same time, for 5 minutes. Added ice cold %2 FBS in PBS and spin down at 1000rpm for 5 minues 4C. There were NO PELLET on both ice incubated and RT incubated tubes. I check under microscope with tryphan blue and there were literally NO CELLS! In facss, nothing!
In second experiment, I diluted 10X RBC with autoclaved dH2O and performed the same setup: Added 1mL into the tube and incubated on ICE and RT on 2 separate experiments at the same time, for 5 minutes. Added ice cold %2 FBS in PBS and spin down at 1000rpm for 5 minues 4C. Again,There were NO PELLET on both ice incubated and RT incubated tubes. I check under microscope with tryphan blue and there were literally NO CELLS! In facs, no cells! Just debries.
In 3rd experiment, diluted it with Millipore water. Added 1mL into the tube and incubated on ICE and RT on 2 separate experiments at the same time, for 5 minutes. this time , I added ice cold PBS ONLY and rest was performed as indicated above. There were some cells in FSCxSSC but all seems like monocytes. No lymphocytes! No granulocytes!
In 4th experiment, diluted 10X to 1X both using Millipore and autoclaved dH2O water seperately. I collected quite equal amount from 2 tumor bearing mice. Incubated on RT and ICE but in DARK. Again, There were some cells in FSCxSSC but all seems like monocytes. No lymphocytes! No granulocytes!
All last 2 experiments gave results as in the attachment (exp 3, capture 1- exp 4 capture2). Experiments performed on NovoCyte Quanteon, which normally gives very stable results in cell lines. I am in the same situation with human whole blood btw! tried to stain with 2% BSA things were not changed!
For 2 years, I used eBioscience 1X RBC lysis buffer adding 1 mL after collection blood. Incubated on RT for 5 mins. To stop the reaction added RT PBS and spin down at 1000rpm for min 4C. And result was like in capture 3 (usin BD CANTO II). I have never been in this kind of situation. Have you ever struggle with this?
It is clear that you are lysing your cells. That is where you should focus your attention. I have a question: after you incubate your cells with the diluted RBC lysis buffer how much PBS +/-BSA are you adding to the tube before you spin the cells down? You should dilute the lysed cells at least 10x -i.e., with 10 mL to get back to isotonic conditions.
The amount of lysing solution you are using seems excessive. I used to do blood preps from transplanted mice to determine engraftment by different hematopoietic stem cell sources. Typically, I would first do a partial red cell depletion by diluting the blood in 2 ml 2% dextran then allowing the red cells to sediment by incubating the tubes in a 37oC water bath for 15 minutes, carefully collecting the SN with a transfer pipette, and pellet the cells. Then I would treat the cells with 0.2 mL Gey's hypotonic lysis solution for 5 minutes at RT, dilute with 2 mL PBS/1% BSA, pellet the cells, then stain for FACS. I never had the cell loss you are seeing.
I learned the method from Christa Mueller-Sieburg. I do not remember if the solution was dextran or dextran sulfate. I am certain it would be in her publications from that time [late 80s/early 90s].
Thank you for your answer. For mouse blood sample (assuming collected 200uL) 4uL for a mL of RBC. For spleen, 3ml of rbc + 20uL of pbs.
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