Protocol Flow cytometry

supplies:
• Flow cytometry tubes
• 15 ml sterile conical tubes
• Vials (for preservation of: serum, blood sample, molecular sample)
• 2 Test tube racks (one for tubes, one for Sample)
• Sterile tips
• Sterile plastic Pasteur pipettes.
• marker

Reagents:
• 70% Ethanol.
• Rapid tests for HBV
• Rapid tests for HCV
• Lymphocyte separation media (LSM)
• Phosphate buffered saline sterile
• Ammonium chloride lysis buffer (ACK)
• anti-human CD13 Mab conjugated with PE-CF594
• anti-human CD44 Mab conjugated with phycoerythrin-cyanine5 (PC5)
• anti-human CD45 Mab conjugated with phycoerythrin -texas red
• anti-human CD90 Mab conjugated with PE-CF594
• anti-human CD133 Mab conjugated with Phycoerythrin (PE)
• anti-human EpCAM Mab conjugated with Fluorescein isothiocyanate
(FITC)
• anti-human ICAM Mab conjugated with peridinin chlorophyll-A (PerCP)
• anti-human ALDH Mab conjugated with Fluorescein isothiocyanate (FITC)
• anti-human cd24 Mab conjugated with Fluorescein isothiocyanate (FITC)
• anti-human CK19 Mab conjugated with peridinin chlorophyll-A (PerCP)
• RNA extraction kit

Equipment:
• Variable automatic pipettes.

Apparatus:
• Laminar flow
• Centrifuge
• Flow cytometer
• Thermal cycler
• Real-time PCR
• Refrigerator.
• Deep Freezer – 80° C
Steps: ‫ﻣﺶ ﻟﻠﺮﺳﺎﻟﺔ‬
Collection of blood
Blood samples will be collected under complete aseptic conditions
About 10 ml of blood will be withdrawn from each patient as well as
controls. (3 EDTA tubes)

Test for hepatitis C antibodies (HCV Abs), hepatitis B surface antigen

(HBsAg)
by using rapid tests for HCV, HBV

Sample backup
About 0.5 ml of blood will be saved in a vial

Plasma separation
Centrifuge one of the 3 tubes and isolate the plasma in a vial.

Isolation of mononuclear cells

• mix the LSM by inverting the bottle gently.
• transfer 3 ml of LSM to a 15 ml centrifuge tube.
DON’T FORGET TO LABEL
• Mix EDTA blood in its tube gentely and Carefully layer 2ml blood over
3 ml of LSM (room temperature) in a 15 ml centrifuge tube, creating a
sharp blood-LSM interface. DO NOT MIX DILUTED BLOOD INTO
THE LSM.
LSM and the blood in a ratio of 1:1.5
• Wash any remaining blood in the tube with PBS and layer it carefuuly
over the LSM
• Centrifugation is done at 2000 rpm for 30 min at 15°C.
CHECK THAT THE CENTRIFUGE HAS STARTED SPINNING
• Centrifugation should sediment erythrocytes and polynuclear leukocytes
and band mononuclear lymphocytes above LSM (Bands will be Plasma
layer -----> Mononuclear cell layer -----> LSM layer -----> RBC pellet) a
• Aspirate the top layer of clear plasma to within 2-3 mm above the
lymphocyte layer by Pasteur pipette
• Aspirate carefully in a circular motion the lymphocyte layer plus about
half of the LSM layer below it and transfer it to a centrifuge tube.
• Add BPS to the lymphocyte layer in the centrifuge tube
• Centrifuge for 10 minutes at room temperature (18-25 °C) at a speed
sufficient to sediment the cells without damage (2000 rpm for 10 min at
15°C.)
Washing removes LSM and reduces the percentage of platelets
• The supernatant was discarded and pellet was dislodged .
 • ACK buffer is added to the dislodged pellet, it’s amount according to the
amount of RBCs in the sample (2-4 cm)
• The tube is then kept in the fridge for 7 minutes
Shak the tube every now and then.
• Centrifuge at 2000 rpm for 10 min at 15°C
• The supernatant was discarded and pellet was dislodged
Prepare 4 flow cytometry tubes (1 control and 3 for MAbs)
DON’T FORGET TO LABEL
• Wash the pellet with PBS by re-suspending the pellet in PBS ,
centrifugation, discarding the supernatant and dislodging the pellet
• re-suspending the pellet in 500 ml PBS
Ø 100 ml for RNA extraction (put them in a vial and preserve in the
freezer)
Ø 400 ml for MAbs
• Divide the 400 ml into 4 flow cytomerty tubes
This should be done in the DARK
• Add the monoclonal antibodies according to instructions
• Incubate the tubes for 20 minutes in the fridge (- 4°C)
Shak the tube every now and then.
• Add PBS to each tube to reach final volume of 500 ml
• Shake well and read on flow cytometery

dye Tube 1 Tube 2 Tube 3 Tube 4

PE CD 133 5 ul CD 133 5 ul CD 133 5 ul
PC5/ PerCP CD 44 0.635 ul ICAM 10 ul CK 19 ???
Control
PE-CF594/
CD 90 5ul CD 13 5 ul CD 45 5 ul tube
PE texas red
FITC EpCAM 20 ul ALDH ??? CD 24 5 ul