MULTIPARAMETER IMMUNOFLUORESCENCE Carleton C. Stewart, Ph.D. & Sigrid J. Stewart R ARKLL P aboratory Cancer Institute L OSWE of Flow Cytometry Department of Health, State of New York Elm and Carlton Streets Buffalo, New York 14263 phone: 716-648-5480 fax: 716-648-8806 email: [email protected] ANTIBODY BINDING TO CELLS RPCI LFC THE LAW OF MASS ACTION Ab + Ep Kf AbEp Kr A Ep AbEp Kf = Kr = b
AbEp A Ep b 6 K range is usually ~ 10 f Kr range is usually ~ 10 -3 Ka = Kf/Kr = 106/10-3 = 109 WAYS ANTIBODIES BIND TO CELLS Specific: Fab to epitope Fc to Fc receptor binding is high affinity and saturable Non Specific: binding is low affinity and not saturable Specific Activity is the concentration of bindable antibody to its epitope divided by the protein concentration. SA = [F(ab')2] (protein) Reasons Antibodies do not bind to cells: overconjugation not purified degradation of binding site aggregation STORING OF ANTIBODIES :
Proteases destroy antibodies in: ascitic fluid serum bacteria Use sodium azide Use highly purified albumin or gelatin as carrier Purify antibodies immediately TITERING ANTIBODIES RPCI LFC AMOUNT BOUND SPECIFIC ANTIBODY NON-SPECIFIC ANTIBODY CONCENTRATION 3 g s/n = 2.5 1 g s/n = 2.1 0.3 g s/n = 2.4 0.1 g s/n = 4.1 0.03 g s/n = 4.8 0.01 g s/n = 4.6
0.001 g s/n = 3.2 auto 3 0.003 g s/n = 3.5 5 Signal to Noise TITER 4 3 2 0 1 2 3 4 5 Dilution 6 7
2 10 2 x = 62 fail y = 96 pass 10 10 3 R1 PE-CD4 4 4 N EW BATCH 10 x = 97 pass 1 1 103 FITC-CD3 10 104
1 10 2 10 TC-CD8 3 10 4 D4 102 -CD4 101 BLOCKING IS IMPORTANT INDIRECT IMMUNOFLUORESCENCE STAINING NO BLOCKING Primary Antibody: murine monoclonal antibody Fc Fab Second Antibody: fluoresceinated goat anti-mouse IgG F(ab')2
FcR A epitope B C D ISOTYPE CONTROL- myeloma protein E F G AUTOFLUORESCENCE CONTROL H BLOCKING WITH GOAT IgG goat IgG add Mab Fab add fluoresceinated goat anti-mouse IgG F(ab')2 VERIFICATION OF BLOCK 1. FcR and non-specific binding FL-MAB + PE-mIgG 2. gIgG + FL-MAB + PE-mIgG LOG FLUORESCENCE EFFECT OF BLOCKING ON ANTIBODY BINDING TO MONONUCLEAR CELLS
CELL VOLUME number TOTAL A C B D channel number variation in gamma 1 myeloma protein binding to macrophages 50 45 40 35 30 25 20 15 10 5 0 0 17 39 4 23 mopc myeloma protein
13 21 log fluorescence Second Reagent Quality F(ab)2 of anti IgG anti IgG cell volume DEAD CELLS CAN BE A PROBLEM They bind antibodies non-specifically They masquerade as specific subsets They cause data misinterpretation ANTIBODIES BIND NONSPECIFICALLY TO DEAD CELLS PE-LAMBDA ALL CELLS VIABLE CELLS A B dead cells FL-KAPPA EMA PROCEDURE lysed, washed cells + 5 g EMA 18 cm.
1 2 10 min. WASH, FIX, AND ANALYZE 3 Evaluating Viability with Ethidium Monoazide % dead in gate = 1% % dead = 5% 4 120 10 SSC SSC 50 40 R9 30 30 40 50 60
80 90 100 Forward Scatter Height --> FSC 120 ONE COLOR IMMUNOPHENOTYPING RPCI LFC SINGLE COLOR IMMUNOPHENOTYPING 1. IgG Block MAB wash FL-second antibody F(ab')2 wash 2. IgG Block B-MAB 3. IgG Block FL-MAB wash wash FL-Avidin wash CORRELATED (LIST MODE) DATA ACQUISITION Entry No. 1 2 3 4 5 6 7
8 k Value 80 100 40 20 90 120 100 110 50 75 110 120 Cell Number 1 1 1 1 2 2 2 2 n n n n Parameter FSC SSC Green Red FSC SSC Green Red
FSC SSC Green Red Analysis of List Mode Data A number of cells region A B C forward scatter CD4 fluorescence region B CD4 FLUORESCENCE region C STRATEGIES IN MULTICOLOR FLOW CYTOMETRY RPCI LFC CELLULAR ANTIGENS cytokines structure enzymes Adhesion Metabolic Receptors
courtesy of Jim Bender FLOW CYTOMETRY VS MICROSCOPIC IMAGING s o rt in g c e lls m o le c u lar qu an t it at io n an t ige n c o e xpre s s io n rare c e lls de t e c t io n c e llu lar i n t e rac t io n s pac ial re lat io n s h ips c o m part m e n t aliz at io n s ign al t o n o is e re je c t io n TWO COLOR IMMUNOPHENOTYPING Antibodies labeled with fluorescein Antibodies labeled with phycoerythrin RPCI LFC TWO COLOR IMMUNOPHENOTYPING 1. IgG Block + MAB wash FL- second antibody F(ab')2 IgG Block + PE-MAB wash wash 2. IgG Block + B-MAB + FL-MAB 3. IgG Block + FL-MAB + PE- MAB wash wash PE-Avidin
wash COMBINED INDIRECT AND DIRECT IMMUNOFLUORESCENCE STAINING Primary Antibody: mMAB NO BLOCKING Second Antibody: FGAM PE-mMAB NO BLOCK CD8 + FGAM 21% 6% 49% PE-CD4 VERIFICATION OF BLOCK Second Reagent Block gIgG + MAB wash FL-GAM wash PE-mIgG gIgG + MAB wash FL-GAM wash mIgG + PE-mIgG COMBINED INDIRECT AND DIRECT IMMUNOFLUORESCENCE STAINING Primary Antibody: mMAB BLOCKING Second Antibody: FGAM PE-mMAB CD8 + FGAM
BLOCKED 12% 42% PE-CD4 THREE COLOR IMMUNOPHENOTYPING Antibodies labeled with Fluorescein Antibodies labeled with Phycoerythrin Antibodies labeled with Tandem Complex to Avidin Tandem Complexes are Texas Red or CY 5 coupled to Phycoerythrin er CP is a natural Tandem Complex of peridinin and chlorophyll a protein SINGLE COLOR HISTOGRAMS FL2-CD4 CD4 --> 10 4 TWO COLOR PATTERN 10 3 color 10 2 CD3-CD4- black 1 CD3+CD4- blue
10 2 CD3 --> 10 CD3 3 10 4 10 3 10 4 FOUR COLOR SINGLE LASER IMMUNOPHENOTYPING Antibodies labeled with fluorescein Antibodies labeled with phycoerythrin (PE) Antibodies labeled with PE/Texas Red Antibodies labeled with PE/CY5 or PerCP FOUR COLOR DUAL LASER IMMUNOPHENOTYPING Antibodies labeled with fluorescein Antibodies labeled with phycoerythrin (PE) Antibodies labeled with PE/CY5 or PerCP Antibodies labeled with APC, CY5 or CY7 3
FL1 FL3 FL3 FL2 FL2 FL3 FL1 FL1 FL3 VISUALIZING EACH POPULATION FL4 FL4 FL1 FL3 FL2 FL3 FL3 FL1 FL2 FL4 4 COLOR FL2
3 COLOR 2 COLOR FL1 BIVARIATE DOT PLOTS CAN BE USED TO DISPLAY THE PATTERNS GENERATED BY MULTIPARAMETER DATA. UNDERSTANDING BINARY LOGIC IS USEFUL P1 + + + + + + + + P2 + + + + + + + + P3 + + + + + + + +
P4 + + + + + + + + P1 P2 P3 P4 Note that in binary logic the 1st parameter is sequenced as -/+, two parameters as --/++, 3 parameters as ----/++++, etc.. for as many parameters that are measured. The problem encountered after 3 parameters is...how does one visualize the multiple populations? COLOR PATTERNS USED TO VIEW COEXPRESSION A b 2 Ab3 Ab1 Ab1 ne eg .(-) os.(+) ne g.(-) no s.(+)
A b 1 A b 2 Ab2 neg.(-) neg.(-) pos.(+) pos.(+) Ab3 neg.(-) neg.(-) neg.(-) neg.(-) color grey yellow cyan green Ab1 neg.(-) pos.(+) neg.(-) pos.(+) Ab3 Ab2 neg.(-) neg.(-) pos.(+) pos.(+)
Ab3 pos.(+) pos.(+) pos.(+) pos.(+) color rust blue violet red Boolean Logic Table For Four Color Immunophenotyping R1 R2 R3 R4 bo o le an Ab1 Ab2 Ab3 Ab4 n o t (R1 o r R2 o r R3 o r R4 ) + R1 an d n o t (R2 o r R3 o r R4 ) + R2 an d n o t (R1 o r R3 o r R4 ) + + R1 an d R2 an d n o t (R3 o r R4 ) + R3 an d n o t (R1 o r R2 o r R4 ) + + R1 an d R3 an d n o t (R2 o r R4 ) + + R2 an d R3 an d n o t (R1 o r R4 ) + + + R1 an d R2 an d R3 an d n o t R4 + R4 an d n o t (R1 o r R2 o r R3 )
+ + R4 an d R1 an d n o t (R2 o r R3 ) + + R4 an d R2 an d n o t (R1 o r R3 ) + + + R4 an d R1 an d R2 an d n o t R3 + + R4 an d R3 an d n o t (R1 o r R2 ) + + + R4 an d R1 an d R3 an d n o t R2 + + + R4 an d R2 an d R3 an d n o t R1 + + + + R4 an d R2 an d R3 an d R1 Ab4 is t h e gat in g an t ibo dy c o lo r gre y y e llo w c y an gre e n ru s t blu e v io le t re d gre y y e llo w c y an gre e n ru s t blu e v io le t re d
Types of Antigen Expression B Ab1 Ab1 Ab1 F Ab2 E Ab2 Ab2 D Ab1 C Ab2 Ab2 Ab2 A Ab1 Ab1 INTERPRETING COEXPRESSION 4 10 C
D 1 9 A2 1 10 C D 1 9 3 10 2 10 CD19 --> 3 10 2 10 1 1 10 10
10 3 10 4 3 4 10 1 10 2 CD7 --> CD7 10 3 10 4 THREE OR MORE COLOR IMMUNOPHENOTYPING wash 1. IgG Block + MAB Biotin- second antibody F(ab')2 wash wash IgG Block + FL- MAB + PE-MAB + TC- Avidin 2. IgG Block + FL-MAB + PE-MAB + B-MAB TC-Avidin
wash wash 3. IgG Block + FL-MAB + PE-MAB + TC-MAB wash TC (third color) = PE/TR or PE/CY5 tandem or PerCP STRATEGY FOR SELECTING FLUOROCHROME: EPITOPE DENSITY Low low-intermediate high FLUOROCHROME phycoerythrin, APC tandem, CY5 fluorescein, PerCP STAINING SOP 50 l washed, and blocked* whole blood or bone marrow lyse, centrifuge, decant, blot, and resuspend pellet Add antibody
combination 15 min. on ice *add 10 l mIg (10 mg/ml) to 1 ml washed whole blood. wash, fix, and analyse TANDEM COMPLEX PROPERTIES TO CONSIDER monocyte binding PE-CY5 light sensitivity PE-CY5 batch variation PE-TR and PE-CY5 Non-specific Binding to Monocytes B PerCP-CD4 C SSC CD25 CD25 A PE-CY5-CD4 FSC PE-fluorescence Effect of Light Exposure on PE-CY5 Tandem Fluorescence
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