FLOW CYTOMETRY IN PRECLINICAL TOXICOLOGY/SAFETY ASSESSMENT - Flow Cytometry in Drug Discovery and Development - page 136

After carbon dioxide asphyxiation, rats were exsanguinated via the caudal vena
cava by collecting blood (approximately 10mL per rat) into a 10mL syringe with
an 18 gauge needle containing 200 m L of 10% w/v disodium EDTA. Blood was
processed immediately; else syringes were rocked at room temperature until pro-
cessed ( 15min). EDTA-anticoagulated blood was aliquoted, sans needle, into

50mL conical tubes in 5 mL volumes. Ice-cold ammonium chloride RBC lysis
buffer (40mL/tube) was added and tubes were inverted gently several times to mix.
Samples were incubated at RT for approximately 20min to allow lysis of RBCs,
centrifuged at 200 g for 5min at RT, and supernatants removed, then washed with
20mL PBS. Pellet was resuspended in 5mL of 10% DMSO-RPMI, vortexed, and
samples were aliquoted (1mL) into 4.5 mL cryovials and placed on dry ice for
transport. Upon arrival (same day) samples were transferred to freezer (

<

80 C) for

storage (up to 7 days).

Enrichment and Cytometry. Cryovials were removed from freezer and thawed in
37 C water bath. PBS (2ml) was added to each vial and vortexed. Replicate sample
vials were combined and washed with PBS before addition of antibody cocktail
containing CD3-, CD45R-, CD42d-, and CD11b/c-(Lin)-FITC and CD36-PE. Cells
were incubated on ice in the dark for 20min, washed again, and then resuspended in
2

m m

g/mL HO in AutoMACS running buffer (AB, 400 L). Anti-PE microbeads

L) were added to each sample and incubated at 4 C for 15min with gentle
tilting on Speci-Mix. AB (9.4mL) was added (10mL total volume) and samples were
centrifuged at 300 g for 10 min at 4 C. Supernatants were removed and pellets
resuspended in 500

m

(200

L AB and then separated using the POSSELDS AutoMACS
program for positive selection of rare events. Positive (CD36 þ enriched) and negative
(lineage) fractions were both collected into 12

m

75mm polystyrene tubes and
processed for cell sorting. Collected fractions were centrifuged at 200 g for 5min
at 4 C. CD36 þ enriched fraction was resuspended in 200 L volume and lineage

fraction was resuspended in 1mL volume of PBS w/1% BSA and placed on wet ice
until cell sorting. CD36 þ /Lin /HO þ cells were sorted and reanalyzed to assess
purity (always

m

90%) and then processed for RNA isolation.

6.6.5.2 GeneChip Analysis Appropriate volume of RNA lysis buffer was added to
sort sample and contents transferred to 1.5mL microfuge tube and vortexed
vigorously. Samples were freeze-thawed twice by placing them on dry ice
followed by 37 C water bath and then stored at 80 C until RNA isolation using

Absolutely RNAMicroprep Kit (Stratagene). Samples were concentrated using RNA
Clean-up Kit-5 (Zymo Research). Total RNA from each concentrate was quantified
using a modified version of RiboGreen RNA Quantification Kit Low Range Assay
(Molecular Probes). Quality was assessed by the presence of 18S and 28S ribosomal
bands using Agilent 2100 Bioanalyzer and RNA Pico LabChip Kit (Agilent). RNA
samples were stored at 80 C until GeneChip analysis.

All samples were randomized into two groups and assigned to two separate
scientists for amplification along with one rat liver RNA, serving as a technical
control, per group. All samples were amplified and biotin-labeled using the NuGEN