|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.)