RESPONDER CELL FREQUENCY ASSAY (RCF): LPA ?Limiting Dilution

Monday, May 10, 2004

Description
RESPONDER CELL FREQUENCY ASSAY (RCF): LPA ?Limiting Dilution

Procedure
1
This assay measures the frequency of pathogen-specific memory CD4 cells (1) by
adding a limiting dilution step to a lymphocyte proliferation assay (LPA). In contrast to the
LPA, the RCF is a quantitative measurement. In addition, RCF uses 24 replicates for each
cell dilution, which decreases the technical variability of the assay. RCF has been
previously shown to correlate with the immune status of individuals against herpesviruses
(2).
Separated PBMC are washed, counted and resuspended in RPMI 1640 medium
supplemented with 1% antibiotics and 10% human AB serum (stimulation medium).
Twenty-four replicate cultures containing 100,000, 50,000, 25,000 and 12,500 PBMC per
well are stimulated with CMV and mock infected control antigens for 8 days. On the last
day of culture, cells are pulsed with 3H thymidine 0.25 礐i/ml for 6 h, harvested, and
incorporated radioactivity counted in a scintillation counter. The responder cell frequency
is calculated as described by Henry et al. (3). Responder wells are defined as those whose
cpm exceeds the mean + 3 SD cpm of the control mock antigen-stimulated cultures at the
same cell concentration. The percent non-responder wells is plotted on a log scale against
the number of cells/well plotted on a linear scale and the RCF is interpolated at the 37%
non-responder well frequency. The statistical considerations underlying this method are
discussed in detail in ref 3. RCF is expressed as the mean number of PBMC required to
detect one CMV-specific proliferating cell. The dynamic range of this method is between
1/12,500 and 1/100,000 cells.
2
II. MATERIALS
?5 x 106 PBMC
?RPMI 1640 with glutamine
?Heat-inactivated human AB serum
?Penicillin/ streptomycin antibiotic solution
?CMV or VZV antigen and control
?96-well round-bottom plates
?3H Thymidine (Thy)
3
III. ASSAY PROCEDURE
A. Preparation of Plates (one to several sets of plates can be prepared at one time)
1. Calculate the volume of medium that needs to be prepared: 0.1ml/well x 72 wells x n?br>of antigens (at least 2, one CMV or VZV and one control) x n?of patients.
2. Prepare an adequate volume of 2 x growth medium (2 x GM) represented by RPMI
1640 with glutamine containing 20% human AB serum and 2% antibiotics.
3. Make antigen dilutions in 2 x GM at pre-established optimal concentration of antigen
(this may vary with the antigen batch from 1:50 to 1:200).
4. Dispense 0.1 ml of antigen in each well of rows B through G. Make one plate / antigen
(CMV or VZV or control).
5. Use plates or store them at -20篊 or lower.
B. Assay Set-up and Harvest
1. Make or thaw antigen plates: 2 plates/ patient, one CMV or VZV and one control
antigen.
2. Using PBMC separated with ficoll-hypaque gradients or CPT tubes (washed and
counted) suspend 107 cells in 10 ml of RPMI. This will generate a 106 cells/ml
suspension. Label the tube: 1.
3. Prepare 3 additional tubes containing 4.5, 4, and 3 ml RPMI and label them 2, 3, and 4,
respectively.
4. Transfer 4.5 ml of the 106 cells/ml suspension (tube 1) into tube 2 and mix. This will
generate a 5 x 105 cells/ml suspension.
5. Transfer 4 ml from tube 2 into tube 3. This will generate a 2.5 x 105 cells/ml
suspension.
6. Transfer 3 ml from tube 3 into tube 4. This will generate a 1.2 5 x 105 cells/ml
suspension.
7. Add 0.1 ml of the 106 cells/ml suspension (tube 1) to each well of rows A and B of the
CMV and control antigen plates (48 wells).
4
8. Add 0.1 ml of the 5 x 105 cells/ml suspension (tube 2) to rows C and D of the CMV and
control antigen plates (48 wells).
9. Add 0.1 ml of the 2.5 x 105 cells/ml suspension (tube 3) to each well of rows E and F
of the CMV and/or VZV and control antigen plates (48 wells).
10. Add 0.1 ml of the 1.25 x 105 cells/ml suspension (tube 4) to each well of rows G and H
of the CMV and/or VZV and control antigen plates (48 wells).
11. Incubate plates for 8 days at 37篊 in a 5% CO2, 90% humidified atmosphere.
12. On day 8 (counting the set-up day as day 0) pulse with a 10 礐i /ml Thy solution, 25
祃/well (0.25 礐i/well) and harvest after 6 hours.
C. Calculations
1. Calculate the mean + 3 s.d. cpm of control antigen cultures for each cell concentration.
Responder wells are defined as the CMV/VZV antigen-stimulated wells that have cpm
> mean + 3 s.d. of the corresponding (same cell concentration) mock-stimulated
controls. Nonresponders are defined as wells with cpm mean +3 s.d. of
corresponding controls.
2. Analyze the highest cell concentration cultures. If all the wells are nonresponders, the
RCF is lower than 1: 100,000 cells. Stop. Proceed if there are responder wells.
3. Analyze the lowest cell concentration cultures. If all the wells are responders, the RCF
is higher than 1: 12,500 cells. Stop. Proceed if there are nonresponder wells.
4. Build a plot (preferably computer-generated) of the percent nonresponder wells on a
log scale against the cell number per well on a linear scale. The RCF corresponds to
the number of cells at which 37% of the CMV antigen-stimulated cultures are
nonresponders.

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RESPONDER CELL FREQUENCY ASSAY (RCF): LPA ?Limiting Dilution