BCIC FACS protocol
For antibodies:
FITC ESA
PE CD24
APC CD44
BD# 347197 lot: 25540
BD# 555428 lot: 08894
BD# 559942 lot: 01798
clone: EBA-1 isotype: Ms IgG1, 
clone: ML5 isotype: Ms IgG2a, 
clone: G44-26 isotype: Ms IgG2b, 
Media: the media, the cells of interest usually live in
FACS buffer: PBS with 1 – 3% CS
 Prepare a single cell suspension from the cell line of interest (in media)
 Count the cells and resuspend them at 1x106 cells per mL (media)
 Transfer L cell suspension into FACS tubes (if you feel more comfortable,
you could just stain 150 000 cells; 150L)
 The following stainings are mandatory:
Per cell line:
Unstained sample
Isotype control tube with FITC, PE and APC
Single FITC
Single PE
Single APC
BCIC stained sample
For subsequent samples:
Unstained sample
BCIC stained sample
 Wash with FACS buffer
 Either decant (leaves 100L cells suspension) or remove the supernatant and
resuspend in 100 - 250L FACS buffer
 Prepare a master mix of antibodies and one with isotype with:
10L ESA FITC
5L CD24 PE
5L CD44 APC per sample (adjust if 150 000 cells are stained)
 Add the correspondent antibody into the tubes (see above) and 20L antibody
mix/isotype mix to the BCIC profile tube
 Flick, flick, flick, flick
 Incubate 10min at RT
 Wash twice with ice cold FACS buffer and resuspend in 250L for analysis
At the FACS machine:
 Prepare your sheet that you can see a FSC/SSC (lin mode) dot plot, a FITC/PE dot
plot (to check for auto fluorescence “tail”) and each color as a histogram
This just needs to be done once; you can save it on your USB stick for
further use
 Use the setup mode of the machine to look at your unstained sample
(autofluorescence control) and position the cells of interest somewhere into the
lower/left area of the FSC/SSC dot plot
Here some cell line references:
sum149
FSC: 430, SSC: 380
sum159
FSC: 405, SSC: 470
BT20
FSC: 350, SSC: 450
MDAMB361 FSC: 350, SSC: 480
HCC70
FSC: 365, SSC: 490
 Then check every channel (histogram) and move the population into the area
between 100 and 101 (negative population)
 Check the isotype control tube (unspecific binding control)
 Make sure that the compensation matrix is set to 0
 Now collect 30000 events for all the tubes
Analysis with flowjo:
import your samples of interest
the following steps are done for the compensation:
open the isotype control tube and gate for your cell population (you can name it
compensation populations)
open a dot plot with FSC (x axis) and FITC (y axis)
create a rectangle gate on the population including at least 98% of the cells (name
it FITC neg)
change the y axis to PE and repeat the negative gating (PE neg)
drag and drop the “compensation populations” gate from the isotype control into
the single stained samples
open the single FITC stain and draw a gate on the FITC pos population using a
FITC vs. FCS dot plot
repeat this steps for PE single stained
open → tools → compensation → define new matrix
name your matrix
drag and drop your populations from the samples into the corresponding box of
the matrix (just FITC and PE, because APC does not need to be compensated, if
you work with PerCP also the compensation needs to be done for all colors)
click “compute and apply” and “yes” for all samples
by the mark in front of the samples you can see, that a compensation is set for
each sample
save your matrix (chose the “edit matrix” tab on the top)
set your markers for analysis on compensated samples:
Histogram method:
open you isotype sample and drag and drop your “compensation populations”
gate into any population to generate a copy, rename it to “cells of interest” and
drag and drop it back to the parent and delete it from the subpopulation (if
anybody figures out how to create a gate copy in an easier way, please let me
know...)
open the “cells of interest” population in a histogram window with compensated
FITC on the x axis
create a bifurcated gate such that the negative marker includes at least 98%
change the x axis to compensated PE and repeat the gating
change the x axis to APC and repeat the gating
You now should have a “cells of interest” population with neg and pos
populations for each color (something like this...)
mark all populations and drag and drop them into your BCIC stained sample
drag and drop the CD24- into the CD44+ population and the ESA+ into the just
generated CD24- population
add statistics for frequency of “cells of interest”