Flow cytometry tutorial

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SPECIAL PROTOCOLS

Pedro M.S. Alves, PhD

31 / 07 / 2007

Molecular Biology

1.1 RNA to cDNA synthesis

Use Invitrogen M-MLV Reverse Transcriptase. Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) uses single-stranded RNA or DNA in the presence of a primer to synthesize a complementary DNA strand. This enzyme is isolated from E. coli expressing a portion of the pol gene of M-MLV on a plasmid. The enzyme is used to synthesize first-strand cDNA up to 7 kb.

1.1.1 Method

Use up-to 4 mg of RNA.

• Mix the following components in an 0.5 mL Eppendorf tube:

• 1.0 mL Oligo dT (10 mM).

• 1.0 mL dNTPs (10 mM)

• 11.5 mL total RNA (1 mg)

• x mL dHO to 1 mL.

• TOTAL= 12 mL.

• Five minutes at 65C.

• Cool-down on ice 1 minute.

• Do Quick-spin...

• Add:

• 4 mL 1 strand buffer (5x).

• 2 mL DTT (0.1 M).

• Mix gently...

• Two minutes at 37C.

• Add 1 mL M-MLV RT

• Mix gently

• Fifty minutes at 37C.

• Fifteen minutes at 70C.

• Ice.

• Store at -80C.

1.2 OmniScript RT - QIAGEN

cDNA synthesis from 50 ng up to 2 mg of total RNA. Scalable.

1.2.1 Method

• Thaw all samples on ice

• Quantify RNA content

• Thaw all buffers

• Prepare mix

1 mix:

• Distribute mix and add RNA sample volume

• Vortex

• Incubate 2 hours at 37C

1.3 Polymerase chain reaction

The protocol that follows uses Taq Platinium from Invitrogen. The 10 PCR buffer does not contains MgCl and therefore it was to be added.

1.3.1 Method

• Obtain the number of samples (Positive and negative controls plus unkowns). Add 10% more for volume loss by pipeting.

• Prepare Master mix. ( n times the values of one mix volume).

• In a 2 ml PCR graded Eppendorf add:

• 5 mL of 10x PCR buffer

• 1.5 mL of 50 mM MgCl2

• 1 mL of 100 mM dNTPs

• 0.5 mL of 10 mM Primer A

• 0.5 mL of 10 mM Primer B

• 0.2 mL Taq Platinium (1 unit).

• up to 50 mL of DEPC water

• Mix master mix by inverting carefully the eppendorf.

• place PCR tubes on a 4C rack.

• transfer 48 mL of master mix per tube

• add 2 mL of sample

• close PCR tubes with PCR tube caps.

1.4 Electrophoresis of nucleotides in an agarose gel

1.4.1 Method

• Weight agarose at 1-1.5% in 1x TAE buffer.

• boil agarose

• cooldown agarose and add ethidium bromide.

• drop in the mold set.

1.5 DNA precipitation

1.5.1 Method

1. Add 1:10 volume of Amonium acetate 3M

2. Mix

3. Add 5 x Ethanol p.a.

4. Mix

5. Store at -80oC.

6. Centrifuge 15’ at full speed 4C .

7. Discard Ethanol

8. Wash with Ethanol 70 %

9. Centrifuge 10’ at full speed C .

10.Speed-Vaccum or room-temperature

11. Resolublize in TE buffer or water.

1.6 Bacteria transformation

Method described in Roche Applied Science Manuals.

1.6.1 Method

1. Thaw bacteria on ice.

2. Cold-down 2 mL eppendorfs on ice.

3. Transfer 40 mL of bacteria suspection to cold eppendorfs

4. Add 0.7 mL of 1:10 water diluted b-mercaptoethanol to the bacteria. Incubate 10 minutes on ice. Mix gently every 2 minutes.

5. Add 2 mL of ligation reaction to the bacteria. Mix and incubate 30 minutes on ice.

6. heat-shock at 42C for exactly 45 seconds.

7. place rapidly bacteria on ice for 2 minutes.

8. add 360 mL of warm SOC media.

9. Rock bacteria at 37C for at least 30 minutes.

10. Spread bacteria in LB-Agar plates.

Cellular Biochemistry

2.1 Proteasome digestion

Use of PBMC derived human proteasome from AB+ donors.

2.1.1 Protocol

Protein solubilization All polypeptides should be solubilized in destilated water at a concentration of 20 mM. Otherwise dissolve in 100 % DMSO.

Animal & Cellular Immunology

3.1 Peptide immunization

Peptide immunization in H-2 mice, solution of IFA, CpG and peptide.

3.1.1 Injection preparation

Prepare the double amount of suspension required¹ .

Mix in this order in parafin sealed 2 mL seringe without emboly:

• 50 mL PBS sterile.

• 50 mg peptide from a 10 mg/mL solution (tap to mix).

• 50 mg CpG (-20C).

• 50 mL IFA. (tap to mix, do not pipet).

3.1.2 Sonication and seringe loading

• Keeping sample solution on ice, sonicate 30 seconds at continous rate, 20% strength. Check if the suspension becomes thick.

• transfer solution from the 2 ml seringe to a 1 mL seringe connected to a hipodermic needle (orange).

• push the emboly to have the peptide solution ready for injection.

• place seringe on ice.

3.1.3 Mice immunization

• Immobilize mice in the hood.

• Inject 100 mL of solution at the base of the tale, intradermically, by introducing the needle in the tale at around half-centimeter from the hair limit.

• Remove slowly the needle to avoid that solution comes out.

3.2 Intracellular IFN-gamma staining

This procedure describes the intracellular staining using flurochrome labelled cells.

3.2.1 Protocol

• after doing superficial labelling with antibody or tetramer, wash cells extensively with PBS.

• Fix cells at 4oC during 10 minutes.

• wash cells with PBS 0.2% BSA, 0.2% SAPONIN

• incubate the monoclonal antibody in the PBS BSA SAPONIN for 30 minutes at 4oC.

• Wash cells with PBS BSA SAPONIN

• Fix cells again.

• Acquisition.

3.3 Tetramer labelling of human T cell lymphocytes

• wash cells with PBS.

• incubate cells tetramer in PBS 0.2% BSA, 50 mM EDTA and incubate for 60 minutes at room temperature. Agitate tubes every 30 minutes.

• wash cells with PBS 0.2% BSA, 50 mM EDTA.

• incubated with anti-human CD8 antibody at 4oC 30 during minutes.

• wash cells extensively in PBS 0.2% BSA, 50 mM EDTA.

• ressuspend in PBS 0.2% BSA, 50 mM EDTA and add 1 mg/mL of Propidium Iodide (PI).

• immediately acquire.

3.4 Tetramer labelling of mouse lymphocytes

For the ex vivo monitoring of T cell responses after peptide immunization.

• Collect 100 mL of blood in eppendorfs containing 7 mL of EDTA solution² .

• Tetramer staining for 45 minutes at r.t..

• To 80 mL of blood add 20 mL of 24G2 (Fc block).

• Add tetramer volume (1-2 mL).

• Wash cells 3 with PBS 3% FCS.

• Incubate cells with CD8-FICT (1:50), CD62-FITC (1:50) in 20 mL of PBS 3% FCS. 20 minutes at 4oC.

• Wash cells 3 with PBS 3% FCS.

• Lysate red blood cells

• prepare fresh lysis buffer (1:10 from stock³ in water).

• incubate cells in 300 mL of lysis buffer.

• wait 3’-5’ until solution becomes clear

• Wash cells 2 with PBS 3% FCS.

• Acquision

3.5 Cytotoxic assay

3.5.1 Targets preparation

• Harvest targets

• wash cells in RPMI 0.1% BSA.

• incubate 510–10 cells in 100 mL of RPMI 0.1% BSA plus 100 mL of fresh NaCr.

• mix and incubate for 60 minutes at 37oC.

• wash cells 3  with RPMI 10% FCS.

• count the target cells and plate in RPMI 0.1% BSA a 96 V-bottom plate (usually 1000 target cells/well).

• prepare at least three wells for minimum⁴ and maximum release ⁵ .

3.5.2 Effector preparation

• Count cells and ressuspend them in RPMI 10% FCS.

• plate cells at the corresponding effector : target ratios.

• final volume / well should be 200 mL in RPMI 0.1% BSA.

3.5.3 Special applications

Lytic efficiency assays Targets are incubated with effectors in presence of increasing concentrations of peptide. Usually targets are incubated after adding peptide solution to the plate (volume = 50 mL).

3.6 ELISA

This section describes a home made protocol to do and develop ELISA detecting mouse IFN-g from co-culture supranatants.

3.6.1 Materials and reagents

• PBS 0.1% Tween 20

• PBS 1% BSA

• ELISA plates (...)

• Detection antibody (to coat plates) anti-mouse IFN-g

• Biotinlated anti-mouse IFN-g.

• Streptavidin

• Development substract

• HSO 4N to stop reaction

• ELISA plate reader

3.6.2 Method

1. On the day -1, coat ELISA plates with detection antibody in PBS.Current antibody is diluted at 1:50 in PBS. Split 50 mL per well. Incubate overnight.

2. On Day 0, discard volume and wash plates 3  with PBS 0.1% Tween 20. Dry out plates tapping on absorbent paper before proceeding.

3. Block with PBS 1 % BSA. 50 mL per well during 2 hours at 37C.

4. Wash plates 3 x with PBS 0.1% Tween 20. Dry out plates tapping on absorbent paper before proceeding.

5. Add culture supranatants (50 mL per well) and standards. Incubate overnight.

6. Wash plates 3 x with PBS 0.1% Tween 20. Dry out plates tapping on absorbent paper before proceeding.

7. Incubate with anti-mouse IFN-g biotinilated antibody in PBS 1% BSA. Add 50 mL per well. Incubate 2 hours at 37C.

8. Wash plates 3 x with PBS 0.1% Tween 20. Dry out plates tapping on absorbent paper before proceeding.

9. Incubate with Streptavidin HRP in PBS at 1:5000. Incubate 1 hour at 37C.

10.Wash plates 3 x with PBS 0.1% Tween 20. Dry out plates tapping on absorbent paper before proceeding.

11.incubate with OPD (o-Phenylenediamine Dihydrocloride). Prepare by dissolving gold and silver tablets in 20 mL of water. Add 50 mL per well.

12.incubate 15 minutes on a dark environment.

13.Stop reaction with 50 mL per well of HSO 4N.

14.Read plates at 492 nm.