Double posting! (2nd part: SP Profiling in Stem Cells)

Up next is my introduction to my Major Project (MP), which is related to Side Population (SP).

Short Introduction

Side population is a defined population of cells that could be distinctly identified from the rest of the heterogeneous cell population with the use of Hoechst 33342 dye in the flow cytometers. Using defined filters that allow the collection of the emission profile of the used dye does this.


The primary objective of my project is to identify the best blockers blocker and its optimal concentration in blocking ATP-binding cassette (ABC) family of transporters. The blockers are Verapamil, Fumitremorgin C and Reserpine respectively.

We will be using bone marrow cells.

ABC family of transporters is responsible for the high efflux of the Hoechst dye that contributes to the low Hoechst staining which could be identified with the use of flow cytometers. We use isolated bone marrow from murine species for SP identification.

Below is a diagram that shows how SP cells are identified and gated.








The following is the protocol in which my laboratory follows to acquire SP profiling in flow cytometry.

(The protocol is only for understanding purposes; so certain information is left out, please understand)


DMEM = Dulbecco’s Modified Eagle’s Medium

HBSS = Hank’s buffer salt solution (Hank’s Balanced Salt Solution)

Protocol (Flow chart):

Euthanize the mice/rodent

Use surgical instruments to remove the fur from the fore and hind limbsRemove and store the fore and hind limbs in cold DMEM+(with EDTA)
Transport the specimens on ice and process in the laminar flow hood
Remove the meat from the fore and hind limbsCut the ends of the femur, tibia, humeral and radius

Flush the interior of the bone until it turns white
Filter the cell suspension obtained with a 100um cell strainer
Centrifuge the cell suspension
Remove all but 500ul of the supernatant
Add erythrocyte-lysing solution in a ratio of 11:1

Incubate for 5 minutes under room temperature
Centrifuge the cell suspension
Remove the supernatant and wash 2 times with cold HBSS+
After washing, centrifuge the cell suspension again
Remove the supernatant and add 2ml of cold DMEM+(without EDTA)

Count the number of cells using a hemocytometer
Calculate the total number of cells in the cell suspension

Prepare and label empty 15ml falcon tubes

(The tubes were labeled “unstained”, “PI only” and “Hoechst + PI”, “Hoechst + blocker + PI”)

Pipette in 1mL of pre-warmed DMEM+(without EDTA) in each of the tubes
Calculate the amount of cell suspension to add to achieve a final concentration of 106 cells/ml for each of the tubes
Place the tubes in the waterbath (37oC)
Add blockers to “Hoechst + blocker + PI” tubes according to the blockers to be used

Pre-incubate the cell suspension for 15mins at 37oC
After pre-incubation, add in the Hoechst 33342 dye to all the tubes and incubate for a further 90mins at 37oC

(Except for “unstained” and “PI only” tubes)
Mix the cell suspension at every 20mins interval during the incubation
After incubation, centrifuge the cell suspension
Remove the supernatant and resuspend in cold HBSS+ with PIStore on ice and out of light before data acquisition on FACSAriaRecord the SP profile acquired

That's it for my entry this week.

So as to not confuse anyone, the protocol was briefly explained, so feel free to ask me questions if there is any doubts.

Many thanks

Quan Jun

TG02

Group 08

08 September 2008