Thursday Dec. 11, 2014: Performed a CD45 depletion on two EDTA tubes of 7.5 mL whole blood. Tube 1 straight depletion, tube 2 WB spiked with estimated 1000 MCF7 cells. 50 uL of concentrated 650 uL volume of WBC fraction isolated from buffy coat before depletion stored as reference. CD45 depletion performed on remaining faction. Post depletion fractions fixed with 4% formaldehyde on ice for 15 minutes(4 tubes total). (more procedures to come)
Today: Performed flow cytometry to sort for DNA+ cells, i.e. has nucleus and is not debris. Permebilized cells with 50 uL of 10x BD perm/wash into 450 uL volume and added 50 uL 10 ug/mL DAPI for staining nucleus.
Calibrated DNA+ with 1 reference sample. 1 depleted had 1350 nucleated cells. 2 depleted(spiked with 1000 MC7) had no cells. I must have lost the cells in the wash process from the fixation. Everyone says all washes have loss. Karen suggested to use higher RPM wash. Go to 2000 RPM on beckman. She points out I don’t care about damage to cells that already fixed.
My though is I need to develop some technology that allows me to do cell washes without loss. I have some ideas on how I can use the track etched polycarbonate membranes to do this. More to come.