Antibodies Available for Immunology Flow Cytometry Lab (revised 1999)

No. Specificity Hybridoma Isotype Comments; % Saturation Notes
1 Control M1/69.HK Rat IgG2b Shows unwanted binding; n.d.  
2 CD4 GK1.5 Rat IgG2b Use #A as 2nd Ab; 67%  
3 CD4 H129.19 Rat IgG2a PE-conjugate. See * below.; 50%  
4 CD8 M5/24 Rat IgG2a Use #A as 2nd Ab; ~50%  
5 CD8 53.6 Rat IgG2a PE-conjugate. See * below. 17%  
6 CD11a (LFA-1) M17/7 Rat IgG2b Use #A as 2nd Ab. See below; ~75%  
7 CD18 (LFA-1, CR3) M18/2 Rat IgG2a Use #A as 2nd Ab. See below; ~75%  
8 CD45/1 M1/9.3 Rat IgG2a Use #A as 2nd Ab; 20%  
9 CD45/2 M1/89.18 Rat IgG2b Use #A as 2nd Ab. See below; 33%  
10 IgG, mouse Polyclonal Goat mixed FITC-conjugated;  
11 IgM, mouse Polyclonal Goat mixed PE-conjugated; 33%  
12 ICAM-1, CD54 YN1/1 Rat IgG2a Use #A as 2nd Ab; 67%  
13 MHC Class I, H-2KDL M1/42 Rat IgG2a Use #A as 2nd Ab; 33%  
14 MHC Class II, H-2I-A, I-E M5/114 Rat IgG2b Use #A as 2nd Ab; n.d.  
15 TCR H57-597 Hamster IgG FITC-conjugated; 67%  
16 Thy-1 (mouse T cell marker) M5/49 Rat IgG2a Use #A as 2nd Ab; 4%  
Numbered Abs above are specific for a marker on mouse lymphocytes. Lettered reagents below are for use as "2nd reagents" for indirect staining of numbered, marker-specific Abs which are not directly conjugated with a fluorochrome.
A IgG, rat Polyclonal Goat mixed FITC-conjugated, see below; ~50%  

Colors: FITC emits green light, PE emits red light. Unless indicated in the Comments column, the above antibodies are not directly conjugated with a fluorochrome. Only green (FITC) indirect stain (anti-rat IgG) is available for unconjugated rat monoclonal Abs. If you stain two markers in the same tube, they must use different colors.

% Occupancy: The percentage of receptors on the cell surface which will be occupied by antibody at the dilution provided for your use. n.d. = not determined. Monoclonal antibody binding is well-described by the one-site binding equation, I/Imax = C/(C + Ch), where I is the observed fluorescence intensity, Imax is the intensity at saturation, Ch is the concentration of antibody which half-saturates, and C is the concentration applied. This equation has been fitted to I vs. C data for most of these antibodies, thereby determining Imax and Ch, and allowing the % occupancy to be calculated at a given C. In two-color samples, green fluorescence intensities exceeding 100 times autofluorescence make it difficult to get accurate red fluorescence readings (the compensation subtraction of green spill-up into the red channel fails). Therefore, green-reported antibodies specific for very high-density receptors (e.g. Thy-1, CD45) are intentionally diluted to give a low % saturation.

* If you use a rat PE conjugate in the same tube with an unconjugated rat Ab to a different marker, the PE conjugate must be applied after the 1st rat Ab and 2nd FITC anti-rat Ab, as a 3rd cycle. This avoids unwanted green staining of the red rat PE conjugate by the green anti-rat 2nd Ab.

Use as 2nd Ab (indirect stain) for all unconjugated rat monoclonal 1st Abs. Absorbed with mouse Ig, so will not stain mouse B cell Ig by cross reaction.

Although 100% of splenocytes are positive with these antibodies, they stain T and B cells with different intensities. (The other antibodies above which stain 100% of splenocytes stain them all with a uniform intensity, namely the other CD45 and MHC class I.)